Anti-pad4 autoantibodies as clinical response biomarkers for the treatment of rheumatoid arthritis

ABSTRACT

The present disclosure relates to the use of anti-PAD4 autoantibodies as a clinical biomarker for rheumatoid arthritis (RA) treatment. The disclosure further provides an assay to detect anti-PAD4 autoantibodies, assay kits for the detection of anti-PAD4 autoantibodies, as well as computer implemented diagnostic methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. application Ser.No. 16/304,055, filed Nov. 21, 2018, which is a U.S.C. § 371 NationalStage Application of International Application No. PCT/EP2017/062479,filed on May 23, 2017, which claims priority to U.S. provisional patentapplication Ser. No. 62/340,560, filed on May 24, 2016, the entirety ofeach of which is hereby incorporated by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in xml(Name: KPL-033US2.xml; Size: 20 KB; and Date of Creation: Feb. 14, 2023)filed with the application is incorporated herein by reference in itsentirety.

BACKGROUND

Rheumatoid arthritis (RA) is a chronic autoimmune disease affectingapproximately 1% of the world's population. It is characterized byinflammation and cellular proliferation in the synovial lining of jointsthat can ultimately result in cartilage and bone destruction, jointdeformity and loss of mobility. RA usually causes problems in severaljoints at the same time, often in a symmetric manner. Early RA tends toaffect the smaller joints first, such as the joints in the wrists,hands, ankles and feet. As the disease progresses, joints of theshoulders, elbows, knees, hips, jaw and neck can also become involved.Unlike other arthritic conditions that only affect areas in or aroundjoints, RA is a systemic disease which can cause inflammation inextra-articular tissues throughout the body including the skin, bloodvessels, heart, lungs and muscles. Consequently, RA imposes an importanteconomic burden on society. Considerable data also suggest that RA isassociated with lowered life expectancy.

RA is a heterogeneous disease and there are no approved treatmentoptions that are highly effective in all patients. Currently there is nocure for RA, and treatment is essentially directed towards relievingpain, reducing inflammation, and stopping or slowing joint damage andbone destruction. The current therapeutic approach is to prescribedisease-modifying antirheumatic drugs (DMARDs) early in the condition,as RA patients treated early with such drugs have better outcomes, withgreater preservation of function, less work disability, and smaller riskof premature death.

RA is characterized by the presence of several types of autoantibodies.The most common forms of autoantibody present in RA patients includerheumatoid factor and anti-citrullinated protein antibodies. Based onlimited publications, autoantibodies directed against peptidylargininedeiminase 4 (PAD4) are present in approximately one-third of RApatients. However, there is currently a poor understanding of thefunction and potential pathogenic role that anti-PAD4 autoantibodies mayhave in RA.

New therapeutic options, including mavrilimumab, have the potential toaddress the unmet medical needs of RA patients. Accordingly, a means toidentify RA patients who are likely to have a positive clinical responseto mavrilimumab (e.g., a diagnostic biomarker) could greatly augment theutility of this novel therapeutic. Similarly, a reliable method fordetecting anti-PAD4 autoantibodies would be of great benefit for the RApopulation.

BRIEF SUMMARY

The present disclosure provides assays for determining the level ofanti-PAD4 autoantibodies in a sample. In some embodiments, the assay fordetermining the level of anti-PAD4 autoantibodies comprises allowinganti-PAD4 autoantibodies present in the sample to bind to recombinantPAD4, binding ruthenylated recombinant PAD4 to the anti-PAD4autoantibody bound to PAD4, detecting the level of anti-PAD4autoantibodies, and determining the level of anti-PAD4 autoantibodies inthe sample. In some aspects, the lower limit of quantification (LLOQ) ofanti-PAD4 autoantibodies for the assay is 5000 U/mL. In someembodiments, the assays may be agglutination assays or homogeneousassays.

The present disclosure provides, inter alia, method of treating arheumatoid arthritis (RA) patient comprising administering an antibodyor antigen-binding fragment thereof that specifically binds to humangranulocyte macrophage colony-stimulating factor receptor alpha(GM-CSFRα) to the patient if the level of anti-peptidylargininedeiminase 4 (anti-PAD4) autoantibodies in a sample from the patient isbelow the lower limit of quantification (LLOQ) for the assay. Alsoprovided is a method of treating an RA patient comprising administeringan antibody or antigen-binding fragment thereof that specifically bindsto GM-CSFRα (e.g., mavrilimumab) to the patient if the level ofanti-PAD4 autoantibodies in a sample from the patient is below the LLOQfor the assay, and the patient presents a level of at least one RAbiomarker in a sample taken from the patient which is above or below apredetermined biomarker threshold level, or is above or below thebiomarker level in one or more control samples. In some aspects, asample is obtained from the patient. In some aspects, the sample isobtained from the patient and is submitted for measurement of the levelof anti-PAD4 autoantibodies in the sample.

The present disclosure also provides a method of treating an RA patientcomprising submitting a sample from the patient for measurement of thelevels of anti-PAD4 autoantibodies in the sample; and administering anantibody or antigen-binding fragment thereof that specifically binds toGM-CSFRα (e.g., mavrilimumab) to the patient if the patient's level ofanti-PAD4 autoantibodies in the sample is below the LLOQ for the assay.Also provided is a method of treating an RA patient comprisingsubmitting a sample from the patient for measurement of the level ofanti-PAD4 autoantibodies in the sample; and suspending theadministration of an antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα (e.g., mavrilimumab) to the patient ifthe patient's level of anti-PAD4 autoantibodies in the sample is abovethe LLOQ for the assay.

The instant disclosure also provides a method of treating an RA patientcomprising measuring the level of anti-PAD4 autoantibodies in a sampleobtained from a patient having RA; determining whether the patient'slevel of anti-PAD4 autoantibodies in the sample is above or below theLLOQ for the assay; and advising a healthcare provider to administer anantibody or antigen-binding fragment thereof that specifically binds toGM-CSFRα (e.g., mavrilimumab) to the patient if the patient's level ofanti-PAD4 autoantibodies is below the LLOQ for the assay. Also providedis a method of treating an RA patient comprising measuring the level ofanti-PAD4 autoantibodies in a sample obtained from a patient having RA;determining whether the patient's level of anti-PAD4 autoantibodies inthe sample is above or below the LLOQ for the assay; and advising ahealthcare provider to suspend the administration of an antibody orantigen-binding fragment thereof that specifically binds to GM-CSFRα(e.g., mavrilimumab) to the patient if the patient's level of anti-PAD4autoantibodies is above the LLOQ for the assay.

The present disclosure also provides a method of treating an RA patientcomprising submitting a sample taken from the patient for measurement ofthe level of anti-PAD4 autoantibodies in the sample; and administeringan antibody or antigen-binding fragment thereof that specifically bindsto GM-CSFRα (e.g., mavrilimumab) to the patient if the patient's levelof anti-PAD4 autoantibodies in the sample is below the LLOQ for theassay.

Also provided is a method of treating an RA patient comprisingsubmitting a sample taken from the patient for measurement of the levelof anti-PAD4 autoantibodies in the sample; and suspending theadministration of an antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα (e.g., mavrilimumab) to the patient ifthe patient's level of anti-PAD4 autoantibodies in the sample is abovethe LLOQ for the assay. Also provided is method of determining whetherto treat a patient diagnosed with RA with a therapeutic regimencomprising the administration of an antibody or antigen-binding fragmentthereof that specifically binds to GM-CSFRα (e.g., mavrilimumab),comprising: measuring, or instructing a clinical laboratory to measurethe level of anti-PAD4 autoantibodies in a sample obtained from thepatient diagnosed with RA; and treating, or instructing a healthcareprovider to treat the patient with a therapeutic regimen comprising theadministration of an antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα (e.g., mavrilimumab) if the patient'slevel of anti-PAD4 autoantibodies in the sample is below the LLOQ forthe assay. In some instances, the level of anti-PAD4 autoantibodies inthe sample is determined using the assay of the invention.

The present disclosure also provides a method of determining whether totreat a patient diagnosed with RA with a therapeutic regimen comprisingthe administration of an antibody or antigen-binding fragment thereofthat specifically binds to GM-CSFRα (e.g., mavrilimumab), comprising:measuring, or instructing a clinical laboratory to measure the level ofanti-PAD4 autoantibodies in a sample from the patient diagnosed with RA;and suspending the treatment, or instructing a healthcare provider tosuspend the treatment of the patient with a therapeutic regimencomprising the administration of an antibody or antigen-binding fragmentthereof that specifically binds to GM-CSFRα (e.g., mavrilimumab) if thepatient's level of anti-PAD4 autoantibodies in the sample is above theLLOQ for the assay.

Also provided is a method of selecting a patient diagnosed with RA as acandidate for treatment with an antibody or antigen-binding fragmentthereof that specifically binds to GM-CSFRα (e.g., mavrilimumab)comprising: measuring, or instructing a clinical laboratory to measurethe level of anti-PAD4 autoantibodies in a sample obtained from apatient diagnosed with RA; and treating, or instructing a healthcareprovider to treat the patient with an antibody or antigen-bindingfragment thereof that specifically binds to GM-CSFRα (e.g.,mavrilimumab) if the patient's level of anti-PAD4 autoantibodies in thesample is below the LLOQ for the assay.

The present disclosure also provides a method of selecting a patientdiagnosed with RA as a candidate for treatment with an antibody orantigen-binding fragment thereof that specifically binds to GM-CSFRα(e.g., mavrilimumab) comprising: measuring, or instructing a clinicallaboratory to measure the level of anti-PAD4 autoantibodies in a sampleobtained from a patient diagnosed with RA; and suspending, orinstructing a healthcare provider to suspend the treatment of thepatient with an antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα (e.g., mavrilimumab) or select analternative treatment if the patient's level of anti-PAD4 autoantibodiesin the sample is above the LLOQ for the assay.

In some aspects of the methods disclosed above, the antibody orantigen-binding fragment thereof that specifically binds to GM-CSFRαcomprises mavrilimumab or an antigen-binding fragment thereof. In someaspects, the antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα consists of mavrilimumab or anantigen-binding fragment thereof. In some aspects, the antibody orantigen-binding fragment thereof that specifically binds to GM-CSFRαspecifically binds to the same epitope as mavrilimumab.

In some aspects, the antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα specifically competes with mavrilimumabfor binding to the same epitope. In some aspects, the antibody orantigen-binding fragment thereof that specifically binds to GM-CSFRαcomprises a heavy chain variable region (VH) having the amino acidsequence set forth in SEQ ID NO: 4, and/or a light chain variable region(VL) having the amino acid sequence set forth in SEQ ID NO: 5 These arethe heavy chain and light chain variable regions of mavrilimumab.

In some aspects, the antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα comprises at least one of thecomplementarity determining regions set forth in SEQID NOS: 6-11. Insome aspects, the patient has been treated with one or more additionalDisease-Modifying Anti-Rheumatic Drugs (DMARDs), either before, during,or after administration of an antibody or antigen-binding fragmentthereof that specifically binds to GM-CSFRα. In some aspects, the DMARDis selected from abatacept, adalimumab, azathioprine, chloroquine,hydroxychloroquine, ciclosporin, D-penicillamine, etanercept, golimumab,infliximab, leflunomide, methotrexate, minocycline, rituximab,sulfasalazine, and combinations thereof. In some aspects, the sampleobtained from the patient comprises one or more of whole blood, bloodserum, plasma, or synovial fluid. In some aspects, the sample obtainedfrom the patient is blood serum.

In some aspects, the methods disclosed above further comprisedetermining, submitting a sample from the patient for determination, orinstructing a clinical laboratory to determine the expression level oractivity of one or more additional biomarkers (e.g., biologicalbiomarkers or clinical biomarkers), to determine at least one clinicalstatus marker, or a combination thereof.

In some aspects, the antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα (e.g., mavrilimumab) is administered at afixed dose. In some aspects, the antibody or antigen-binding fragmentthereof that specifically binds to GM-CSFRα (e.g., mavrilimumab) isadministered at a fixed dose of about 30 mg/dose, about 100 mg/dose, orabout 150 mg/dose.

In some aspects, the patient's anti-PAD4 autoantibody level is measuredin an immunoassay. In some aspects, the immunoassay employs detectablylabeled PAD4. In some aspects, the detectably labeled PAD4 isruthenylated PAD4. In some aspects, the immunoassay detectsanti-PAD4-induced PAD4 crosslinking.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows anti-PAD4 autoantibody levels measured in RA subjects fromthree different clinical studies using mavrilimumab (studies A, B, andC).

DETAILED DESCRIPTION

The present disclosure relates to using the presence or absence ofanti-peptidylarginine deiminase 4 (anti-PAD4) autoantibodies in a samplefrom an RA patient as a biomarker to determine the appropriate course oftreatment. For example, whether to administer an antibody orantigen-binding fragment thereof that specifically binds to thegranulocyte macrophage colony-stimulating factor receptor alpha(GM-CSFRα). Accordingly, the disclosure provides methods for diagnosinga patient, methods for treating a patient (e.g., with an antibody suchas mavrilimumab), methods for selecting or non-selecting a patient fortreatment (e.g., with an antibody such as mavrilimumab), selecting acertain treatment (e.g., mavrilimumab), suspending temporarily orpermanently a treatment, determining the prognosis of a patient, ormonitoring the effect of a treatment, wherein those methods comprisedetermining the presence or absence of anti-PAD4 autoantibodies and/orquantifying the level of anti-PAD4 autoantibodies in a sample taken fromthe patient. In certain embodiments, the presence or absence ofanti-PAD4 is determined using the assay taught here.

In order that the present disclosure can be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

I. Definitions

In this specification and the appended claims, the singular forms “a”,“an” and “the” include plural referents unless the context clearlydictates otherwise. The terms “a” (or “an”), as well as the terms “oneor more,” and “at least one” can be used interchangeably herein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

Wherever aspects are described herein with the language “comprising,”otherwise analogous aspects described in terms of “consisting of” and/or“consisting essentially of” are also provided.

The term “about” as used in connection with a numerical value throughoutthe specification and the claims denotes an interval of accuracy,familiar and acceptable to a person skilled in the art. In general, suchinterval of accuracy is ±15%.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Systéme Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects oraspects of the disclosure, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety.

As used herein, the term “antibody” refers to at least the minimalportion of an antibody which is capable of binding to antigen, e.g., atleast the variable domain of a heavy chain (VH) and the variable domainof a light chain (VL) in the context of a typical antibody produced by aB cell. Basic antibody structures in vertebrate systems are relativelywell understood. See, e.g., Harlow et al., Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). Antibodiesor antigen-binding fragments, variants, or derivatives thereof include,but are not limited to, polyclonal, monoclonal, human, humanized, orchimeric antibodies, single chain antibodies, epitope-binding fragments,e.g., Fab, Fab′ and F(ab′)2, Fd, Fvs, single-chain Fvs (scFv),single-chain antibodies, disulfide-linked Fvs (sdFv), fragmentscomprising either a VL or VH domain, fragments produced by a Fabexpression library. ScFv molecules are known in the art and aredescribed, e.g., in U.S. Pat. No. 5,892,019. Immunoglobulin or antibodymolecules encompassed by this disclosure can be of any type (e.g., IgG,IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1and IgA2) or subclass of immunoglobulin molecule. Thus, in view if thisdefinition of the term “antibody,” references to an antibody such asmavrilimumab refers to the mavrilimumab antibody and also toantigen-binding fragments, variants, and derivatives thereof.

By “specifically binds,” it is generally meant that an antibody orfragment, variant, or derivative thereof binds to an epitope via itsantigen-binding domain, and that the binding entails somecomplementarity between the antigen binding domain and the epitope.According to this definition, an antibody is said to “specifically bind”to an epitope when it binds to that epitope via its antigen-bindingdomain more readily than it would bind to a random, unrelated epitope.

An antibody or antigen-binding fragment, variant, or derivative thereofis said to competitively inhibit binding of a reference antibody orantigen-binding fragment to a given epitope if it preferentially bindsto that epitope to the extent that it blocks, to some degree, binding ofthe reference antibody or antigen binding fragment to the epitope.Competitive inhibition can be determined by any method known in the art,for example, competition ELISA assays. A binding molecule can be said tocompetitively inhibit binding of the reference antibody orantigen-binding fragment to a given epitope by at least 90%, at least80%, at least 70%, at least 60%, or at least 50%.

Antibodies or antigen-binding fragments, variants, or derivativesthereof disclosed herein can be described or specified in terms of theepitope(s) or portion(s) of an antigen, e.g., a target polysaccharidethat they recognize or specifically bind. For example, the portion ofGM-CSFR that specifically interacts with the antigen-binding domain ofan anti-GM-CSFR antibody (e.g., mavrilimumab) is an “epitope.”

The term “autoantibody”, as used herein (e.g., to refer to an anti-PAD4autoantibody) refers to an antibody that is produced by the immunesystem of a subject and that is directed against an autoantigen.Autoantibodies may attack the body's own cells, tissues, and/or organs,causing inflammation and damage. As used herein, the term “autoantigen”refers to an endogenous antigen (e.g., PAD4), or an active fragmentthereof, that stimulates the production of autoantibodies in a subject'sbody, as in autoimmune reactions. The term also encompasses anysubstances that can form an antigen-antibody complex with autoantibodiespresent in a subject or in a biological sample obtained from a subject.

The term “treatment” is used herein to characterize a method that isaimed at delaying or preventing the onset of a disease or condition(e.g., RA); slowing down or stopping the progression, aggravating, ordeteriorations of the symptoms of the condition; bringing aboutameliorations or the symptoms of the condition; and/or curing thecondition. A treatment may be administered prior to the onset of thedisease, for a prophylactic or preventive action. It may also beadministered after initiation of the disease, for a therapeutic action.

The terms “subject” or “patient” as used herein refer to any subject,particularly a mammalian subject, for whom diagnosis, prognosis, ortherapy of RA is desired. As used herein, the terms “subject” or“patient” include any human or nonhuman animal. The term “nonhumananimal” includes all vertebrates, e.g., mammals and non-mammals, such asnonhuman primates, sheep, dogs, cats, horses, cows, bears, chickens,amphibians, reptiles, etc.

As used herein, phrases such as “rheumatoid arthritis patient” or “RApatient” includes subjects, such as mammalian subjects, that wouldbenefit from the administration of a therapy, diagnostic procedure, orpreventive treatment for RA. In some aspects, the term “RA patient”refers to a subject that presents one or more symptoms indicative of RA(e.g., pain, stiffness or swelling of joints), or that is screened forRA (e.g., during a physical examination). Alternatively or additionally,the term “RA patient” also encompasses subjects suspected of having RAor who may have one or more risk factors (e.g., age, sex, familyhistory, smoking, etc.). The term encompasses subjects that have notbeen tested for RA as well as subjects that have received an initialdiagnosis.

In some aspects of the present disclosure, a subject is a naïve subject.A naïve subject is a subject that has not been administered a RAtherapy, for example a therapeutic agent such as a DMARD. In someaspects, a naïve subject has not been treated with a therapeutic agentprior to being diagnosed with RA. In another aspect, a subject hasreceived therapy and/or one or more doses of a therapeutic agent (e.g.,a DMARD) prior to being diagnosed as having RA. The therapeutic agentcan be a small molecule drug, an antibody, a fusion protein, acorticosteroid, a NSAID (e.g., ibuprofen, naproxen, etc.), a DMARD(e.g., methotrexate, leflunomide, chloroquine, hydroxychloroquine,azathioprine, cyclosporine, D-penicillamine, sulfasalazine, minocycline,etc.), a biologic agent (e.g., abatacept, adalimumab, anakinra,certolizumab, etanercept, golimumab, infliximab, rituximab, tofacitinib,or mevralimumab), or a combination thereof.

In one aspect, the therapeutic agent used according to methods disclosedherein is an antibody, e.g., an anti-GM-CSFRα antibody such asmavrilimumab. Accordingly, in some aspects, a subject has received or isa candidate to receive at least one therapeutically effective dose of anantibody (e.g., mavrilimumab). A subject can be administered at leastone therapeutically effective dose of an anti-GM-CSFRα antibody if thesubject's anti-PAD4 level is below a predetermined anti-PAD4 thresholdlevel, or if anti-PAD4 autoantibodies are absent or their level is lowrelative to the anti-PAD4 level in one or more control samples. Also, asubject can be deemed eligible to receive at least one therapeuticallyeffective dose of an anti-GM-CSFRα antibody disclosed herein if thesubject's level of anti-PAD4 autoantibodies is below a predeterminedanti-PAD4 autoantibody threshold level, or if anti-PAD4 autoantibodiesare absent or their level is low relative to the anti-PAD4 autoantibodylevel in one or more control samples.

In some aspects, the presence or absence of anti-PAD4 autoantibodies isbased on the lower limit of quantification (LLOQ) for the assay used. Insome embodiments, the assay used is taught here. The bioanalyticalparameter “Lower limit of quantification” or “LLOQ” is defined as“thelowest amount of an analyte in a sample that can be quantitativelydetermined with suitable precision and accuracy” (FDA: Guidance forIndustry, Bioanalytical Method Validation, May 2001).

The terms “normal” and “healthy” when applied to a subject are usedherein interchangeably. They refer to a subject that has not shown anyRA symptoms, and that has not been diagnosed with RA or with cartilageor bone injury. Preferably, a normal subject is not on medicationaffecting RA and has not been diagnosed with any other disease (inparticular an autoimmune inflammatory disease). In certain embodiments,normal subjects may have similar sex, age, and/or body mass index ascompared with the subject from which the biological sample to be testedwas obtained. The term “normal” is also used herein to qualify a sampleobtained from a healthy subject.

The term “therapy” as used herein includes any means for curing,mitigating, or preventing RA, including, for example, therapeuticagents, instrumentation, supportive measures, and surgical orrehabilitative procedures. In this respect, the term therapy encompassesany protocol, method and/or therapeutic or diagnostic that can be usedin prevention, management, treatment, and/or amelioration of RA.

The term “therapeutic agent” as used herein refers to anytherapeutically active substance that is administered to a subjecthaving RA to produce a desired, usually beneficial, effect. The termtherapeutic agent includes, e.g., classical low molecular weighttherapeutic agents commonly referred to as small molecule drugs andbiologics including but not limited to antibodies or active fragmentsthereof, peptides, protein drugs, protein conjugate drugs, etc. Atherapeutic agent can also be a pro-drug, which metabolizes into thedesired therapeutically active substance when administered to a subject.In some aspects, the therapeutic agent is a prophylactic agent. Inaddition, a therapeutic agent can be pharmaceutically formulated.

A “therapeutically effective” amount as used herein is an amount oftherapeutic agent that provides some improvement or benefit to a subjecthaving RA. Thus, a “therapeutically effective” amount is an amount thatprovides some alleviation, mitigation, and/or decrease in at least oneclinical symptom of RA. Clinical symptoms associated with RA that can betreated by the methods and systems of the disclosure are well known tothose skilled in the art. Further, those skilled in the art willappreciate that the therapeutic effects need not be complete orcurative, as long as some benefit is provided to the subject.

As used herein, a “sufficient amount” or “an amount sufficient to”achieve a particular result in a patient having RA refers to an amountof a therapeutic agent (e.g., an antibody such as mavrilimumab) that iseffective to produce a desired effect, which is optionally a therapeuticeffect (i.e., by administration of a therapeutically effective amount).

The term “biological sample” is used herein in its broadest sense. Abiological sample is generally obtained from a subject. A sample may beof any biological tissue or fluid with which biomarkers of the presentdisclosure may be assayed. Frequently, a sample will be a “clinicalsample”, i.e., a sample derived from a patient. Such samples include,but are not limited to, bodily fluids which may or may not containcells, e.g., blood (e.g., whole blood, serum or plasma), urine, synovialfluid, saliva, tissue or fine needle biopsy samples, and archivalsamples with known diagnosis, treatment and/or outcome history.Biological samples may also include sections of tissues such as frozensections taken for histological purposes. The term “biological sample”also encompasses any material derived by processing a biological sample.Derived materials include, but are not limited to, proteins extractedfrom the sample. Processing of a biological sample may involve one ormore of: filtration, distillation, extraction, concentration,inactivation of interfering components, addition of reagents, and thelike. In some aspects, the biological sample is a serologic sample andis (or is derived from) whole blood, serum or plasma obtained from asubject.

As used herein, the term “control”, when used to characterize a subject,refers to a subject that is healthy or to a patient who has beendiagnosed with a specific disease other than RA. The term “controlsample” refers to one, or more than one, biological sample that has beenobtained from a healthy subject or from a patient diagnosed with adisease other than RA.

In order to apply the methods and systems of the disclosure, samplesfrom a patient can be obtained before or after the administration of atherapy to treat RA. In some cases, successive samples can be obtainedfrom the patient after therapy has commenced or after therapy hasceased. Samples can, for example, be requested by a healthcare provider(e.g., a doctor) or healthcare benefits provider, obtained and/orprocessed by the same or a different healthcare provider (e.g., a nurse,a hospital) or a clinical laboratory, and after processing, the resultscan be forwarded to the original healthcare provider or yet anotherhealthcare provider, healthcare benefits provider or the patient.Similarly, the determination of the presence or absence of biomarker(e.g., absence or presence of anti-PAD4 autoantibodies) and/orquantification of level of the biomarker (e.g., quantification of theamount of anti-PAD4 autoantibodies), comparisons between levels ofbiomarker, evaluation of the absence/presence or levels of biomarker,and treatment decisions, can be performed by one or more healthcareproviders, healthcare benefits providers, and/or clinical laboratories.

As used herein, the term “healthcare provider” refers to individuals orinstitutions that directly interact and administer to living subjects,e.g., human patients. Non-limiting examples of healthcare providersinclude doctors, nurses, technicians, therapists, pharmacists,counselors, alternative medicine practitioners, medical facilities,doctor's offices, hospitals, emergency rooms, clinics, urgent carecenters, alternative medicine clinics/facilities, and any other entityproviding general and/or specialized treatment, assessment, maintenance,therapy, medication, and/or advice relating to all, or any portion of, apatient's state of health, including but not limited to general medical,specialized medical, surgical, and/or any other type of treatment,assessment, maintenance, therapy, medication and/or advice.

As used herein, the term “clinical laboratory” refers to a facility forthe examination or processing of materials derived from a livingsubject, e.g., a human being. Non-limiting examples of processinginclude biological, biochemical, serological, chemical,immunohematological, hematological, biophysical, cytological,pathological, genetic, or other examination of materials derived fromthe human body for the purpose of providing information, e.g., for thediagnosis, prevention, or treatment of any disease or impairment of, orthe assessment of the health of living subjects, e.g., human beings.These examinations can also include procedures to collect or otherwiseobtain a sample, prepare, determine, measure, or otherwise describe thepresence or absence of various substances in the body of a livingsubject, e.g., a human being, or a sample obtained from the body of aliving subject, e.g., a human being.

As used herein, the term “healthcare benefits provider” encompassesindividual parties, organizations, or groups providing, presenting,offering, paying for in whole or in part, or being otherwise associatedwith giving a patient access to one or more healthcare benefits, benefitplans, health insurance, and/or healthcare expense account programs.

In some aspects, a healthcare provider can administer or instructanother healthcare provider to administer a therapy to treat RA. Ahealthcare provider can implement or instruct another healthcareprovider or patient to perform the following actions: obtain a sample,process a sample, submit a sample, receive a sample, transfer a sample,analyze or measure a sample, quantify a sample, provide the resultsobtained after analyzing/measuring/quantifying a sample, receive theresults obtained after analyzing/measuring/quantifying a sample,compare/score the results obtained after analyzing/measuring/quantifyingone or more samples, provide the comparison/score from one or moresamples, obtain the comparison/score from one or more samples,administer a therapy (e.g., a therapeutic agent that treats RA),commence the administration of a therapy, cease the administration of atherapy, continue the administration of a therapy, temporarily interruptthe administration of a therapy, increase the amount of an administeredtherapeutic agent, decrease the amount of an administered therapeuticagent, continue the administration of an amount of a therapeutic agent,increase the frequency of administration of a therapeutic agent,decrease the frequency of administration of a therapeutic agent,maintain the same dosing frequency on a therapeutic agent, replace atherapy or therapeutic agent by at least another therapy or therapeuticagent, combine a therapy or therapeutic agent with at least anothertherapy or additional therapeutic agent.

In some aspects, a healthcare benefits provider can authorize or deny,for example, collection of a sample, processing of a sample, submissionof a sample, receipt of a sample, transfer of a sample, analysis ormeasurement a sample, quantification a sample, provision of resultsobtained after analyzing/measuring/quantifying a sample, transfer ofresults obtained after analyzing/measuring/quantifying a sample,comparison/scoring of results obtained afteranalyzing/measuring/quantifying one or more samples, transfer of thecomparison/score from one or more samples, administration of a therapyor therapeutic agent, commencement of the administration of a therapy ortherapeutic agent, cessation of the administration of a therapy ortherapeutic agent, continuation of the administration of a therapy ortherapeutic agent, temporary interruption of the administration of atherapy or therapeutic agent, increase of the amount of administeredtherapeutic agent, decrease of the amount of administered therapeuticagent, continuation of the administration of an amount of a therapeuticagent, increase in the frequency of administration of a therapeuticagent, decrease in the frequency of administration of a therapeuticagent, maintain the same dosing frequency on a therapeutic agent,replace a therapy or therapeutic agent by at least another therapy ortherapeutic agent, or combine a therapy or therapeutic agent with atleast another therapy or additional therapeutic agent.

In addition a healthcare benefits provider can, e.g., authorize or denythe prescription of a therapy, authorize or deny coverage for therapy,authorize or deny reimbursement for the cost of therapy, determine ordeny eligibility for therapy, etc.

In some aspects, a clinical laboratory can, for example, collect orobtain a sample, process a sample, submit a sample, receive a sample,transfer a sample, analyze or measure a sample, quantify a sample,provide the results obtained after analyzing/measuring/quantifying asample, receive the results obtained afteranalyzing/measuring/quantifying a sample, compare/score the resultsobtained after analyzing/measuring/quantifying one or more samples,provide the comparison/score from one or more samples, obtain thecomparison/score from one or more samples, or other related activities.

The terms “biomarker” and “marker” are used herein interchangeably. Theyrefer to a predictive factor that is a distinctive indicator of abiological process, biological event, and/or pathologic condition. Asused herein, the term biomarker encompasses both clinical markers andbiological markers. Thus, in the context of the present invention, theterm “biomarker” encompasses anti-PAD4 autoantibodies as well as otherRA “biological biomarkers” comprising proteins such as calpastatin,BRAF, C-reactive protein, serum amyloid A, interleukin 6, S100 proteins,osteopontin, rheumatoid factor, matrix metalloprotease 1, matrixmetalloprotease 3, hyaluronic acid, sCD14, angiogenesis markers,products of bone, cartilage, or synovium metabolism, EYA4, PDZD2, TNF inblood or synovium, IL-1b, IL-17, rheumatoid factor (RF),anticitrullinated peptide antibodies (ACPA), and combinations thereof.The biological markers disclosed herein include also the genes encodingthose proteins (DNA and/or RNA), as well as metabolic products.

The terms “labeled”, “detectably labeled,” “labeled with a detectableagent,” and “labeled with a detectable moiety” are used hereininterchangeably. These terms are used to specify that an entity (e.g.,full length PAD4 or a PAD4-peptide targeted by autoantibodies) can bevisualized, for example, following binding to another entity (e.g., ananti-PAD4 autoantibody). Preferably, a detectable agent or moiety isselected such that it generates a signal which can be measured and whoseintensity is related to the amount of bound entity. In array-basedmethods, a detectable agent or moiety is also preferably selected suchthat it generates a localized signal, thereby allowing spatialresolution of the signal from each spot on the array. Methods forlabeling proteins and polypeptides are well-known in the art. Labeledpolypeptides, e.g., labeled PAD4 or fragments thereof, can be preparedby incorporation of or conjugation to a label, that is directly orindirectly detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical, or chemical means, or any othersuitable means. Suitable detectable agents include, but are not limitedto, various ligands, radionuclides, fluorescent dyes, chemiluminescentagents, microparticles, enzymes, colorimetric labels, magnetic labels,and haptens. In some aspects of the present disclosure, recombinant PAD4or fragments thereof can be labeled with ruthenium to yield luminescentRu(II) metal complexes.

The term “mavrilimumab” refers to a human IgG4 monoclonal antibodydesigned to modulate macrophage activation, differentiation and survivalby targeting GM-CSFRα as described, for example, in U.S. Pat. Nos.8,263,075 and 8,506,960, and U.S. Publ. Nos. US20090130093,US20120141464, and US20140079708, all of which are herein incorporatedby reference in their entireties. US20140335081 describes the use ofmavrilimumab to treat RA. Mavrilimumab, as used herein, encompasses themavrilimumab antibody comprising a heavy chain of SEQ ID NO: 2 and alight chain of SEQ ID NO: 3, as well as antigen-binding fragmentsthereof.

Mavrilimumab is a potent neutralizer of the biological activity ofGM-CSFRα and, without wishing to be bound by theory, may exerttherapeutic effects by binding GM-CSFRα on leukocytes within thesynovial joints of RA patients, leading to reduced cell survival andactivation. U.S. Pat. No. 8,263,075 reports the isolation andcharacterization of mavrilimumab and variants of it which share anability to neutralize the biological activity of GM-CSFRα with highpotency. The functional properties of these antibodies are believed tobe attributable, at least in part, to binding a Tyr-Leu-Asp-Phe-Glnmotif at positions 226 to 230 of human GM-CSFRα (this sequence of aminoacids is set forth in SEQ ID NO: 12), thereby inhibiting associationbetween GM-CSFRα and its ligand GM-CSF. The amino acid sequences of themavrilimumab heavy chain (HC; set forth in SEQ ID NO: 2); light chain(LC; set forth in SEQ ID NO:3); heavychain variable region (VH; setforth in SEQ ID NO:4); light chain variable region (VL; set forth in SEQID NO:5); heavy chain variable region complementarity determiningregions (VH-CDR1, VH-CDR2, and VH-CDR3, set forth in SEQ ID NOs: 6, 7and 8, respectively), and light chain variable region CDRs (VL-CDR1,VL-CDR2, and VL-CDR3; set forth in SEQ ID NOs: 9, 10, and 11respectively).

The term “PAD4” as used herein refers to peptidyl arginine deiminase,type IV and isoforms thereof. The amino acid sequence of a human PAD4 isfound in Uniprot: Q9UM07; and set forth in SEQ ID NO:13). In humans thisprotein is encoded by the PADI4 gene, and fragments thereof (Jones etal. Curr Opin Drug Discov Devel. 12: 616-627 (2009)). Criticalautoantibodies in RA are directed at citrullinated residues on differentproteins such as fibrin, filaggrin, fibronectin, enolase and vimentin.Citrullinated proteins are generated by the post-translationalconversion of arginine residues to citrulline, a process catalyzed bycalcium-dependent peptidyl arginine deiminases (PAD)s.

PAD4 is widely believed to play a role in RA disease onset andprogression because RA-associated mutations in the PAD4 gene have beenidentified in a variety of populations. See, for example, Suzuki et al.,Nat. Genet. 34: 395-402 (2003); Iwamoto et al., Rheumatology 45: 804-807(2006); Harney et al., Rheumatology 44: 869-872 (2005); and Cantaert etal., Ann. Rheum. Dis. 64: 1316-1320 (2005). PAD4 is not only involved inthe generation of citrullinated epitopes, it is in itself a target forRA-specific antibodies. Autoantibodies against PAD4 have been describedin RA patients. See, for example, Takizawa et al., Scand. J. Rheumatol.3: 212-215 (2005); Roth et al., Clin. Exp. Rheumatol. 1: 12-18 (2006);Halvorsen et al., Ann. Rheumatol. Dis. 67: 414-417 (2008); and Zhao etal., J. Rheumatol., 35: 969-974 (2008).

The term “PAD3” as used herein refers to the protein arginine deiminasetype 3, and isoforms thereof. The amino acid sequence of a human PAD3 isfound in Uniprot Q9ULW8. This protein is encoded in humans by the PADI3gene.

The term “GM-CSFR” as used herein refers to granulocyte-macrophagecolony stimulating-factor (GM-CSF) receptor. GM-CSFR is a member of ahighly conserved cytokine receptor super family. GM-CSFR comprises twosubunits which result in different affinities for GM-CSF observed onsome hematopoietic cells. The first subunit is commonly referred to asthem subunit, and is an 85 Kd protein which can bind GM-CSF by itselfwith low affinity. Multiple other isoforms of the GM-CSFRα chain, somemembrane-bound and some soluble, have been described, however the alisoform appears to be the predominant form expressed on the cell surfaceof neutrophils and macrophages (Crosier et al., Br J Haematol.98:540-548 (1997)). The extracellular portion of GM-CSFR α₁ is highlyglycosylated. The receptor has a second subunit, the β chain, which doesnot bind to GM-CSF by itself. Rather, it binds GM-CSF when associatedwith the α₁ receptor. The terms “GM-CSFRa,” “GM-CSFRα,” “GM-CSFR alpha”and grammatical variants thereof refer to the α₁ subunit of GM-CSFR,this protein may have the amino acid sequence set forth in SEQ ID NO: 1.

II. Anti-PAD4 Autoantibodies as a Clinical Response Biomarker

RA is characterized by the presence of several types of autoantibodies.Autoantibodies directed against PAD4 are present in approximatelyone-third of RA patients. However, there is currently a poorunderstanding of the function and potential pathogenic role theanti-PAD4 autoantibodies may have in RA. New therapeutic options,including therapeutic antibodies that specifically bind to humanGM-CSFRα such as mavrilimumab are important to address the unmet medicalneeds of RA patients. Means to identify RA patients likely to have agood clinical response to these antibodies, i.e., a diagnostic orclinical response biomarker, could greatly augment the utility of thisnovel therapeutic.

We have found that the presence of anti-PAD4 autoantibodies in the serumof RA subjects prior to the initiation of therapy with mavrilimumab is apredictor of clinical response. Anti-PAD4 autoantibody levels have beenmeasured in serum using a novel assay that detects anti-PAD4 antibodiesby their ability to bind PAD4. This assay was used to quantify anti-PAD4autoantibody levels in the baseline serum samples of the RA subjectsentered into a clinical study. Statistical analysis showed that RAsubjects with anti-PAD4 autoantibody levels below the lowest limit ofquantification (LLOQ) of the assay had a significantly increasedclinical response to mavrilimumab compared to placebo, while theresponse to mavrilimumab by subjects with detectable anti-PAD4autoantibody levels was more comparable to the response to placebo. Thisfinding supports the utility of testing for the presence of anti-PAD4autoantibodies prior to initiation of mavrilimumab therapy to identifypatients who will have a positive clinical outcome if treated withmavrilimumab compared to DMARD standard-of-care therapy. In addition,there are currently no diagnostic tests employed by rheumatologists toaid in the selection of therapy options to treat RA patients. Measuringanti-PAD4 autoantibody levels (through the blood test) to indicate thata patient is likely to have a much better outcome if treated withmavrilimumab than if treated with DMARDs alone is a significant advancein the treatment paradigm for this disease. In some embodiments, theanti-PAD4 autoantibody levels are measured using the novel method taughthere.

The term “level”, e.g., as in “anti-PAD4 autoantibody level,” “anti-PAD4autoantibody level,” “level of anti-PAD4 autoantibodies,” “patient'slevel of anti-PAD4 autoantibodies,” and grammatical variants thereofrefers to a measurement that is made using any analytical method fordetecting presence or expression of anti-PAD4 autoantibodies in abiological sample and that indicates the presence, absence, absoluteamount or concentration, relative amount or concentration, titer,expression level, ratio of measured levels, or the like, of, for, orcorresponding to anti-PAD4 autoantibodies in the biological sample. Theexact nature of the “value” or “level” depends on the specific designsand components of the particular analytical method employed to detectanti-PAD4 autoantibodies.

As used herein with respect to the detection of anti-PAD4 autoantibodiesin a sample obtained from a subject, the terms “absent” or “present”refers to whether the level of anti-PAD4 autoantibodies is below orabove the lowest limit of quantification (LLOQ) for the analyticalmethod used to detect anti-PAD4 autoantibodies in the biological sample

As used herein with reference to anti-PAD4 autoantibodies, the terms“elevated levels,” “high levels,” and variants thereof refer to a levelin a biological sample (e.g., blood serum) that is higher than a controllevel or range. Thus, the level measured in a particular biologicalsample can be compared with level or range of levels determined insimilar control samples. In some aspects, the control sample would be asample obtained from a subject with no detectable RA symptoms. In otheraspects, the control sample would be a sample obtained from a subjectwho is seropositive for anti-PAD4 autoantibodies (e.g., a subject with alevel of anti-PAD4 autoantibodies in serum that has been determined tobe unresponsive to therapy with mavrilimumab). The level of anti-PAD4autoantibodies is said to be elevated wherein the anti-PAD4autoantibodies are present in the test sample at a higher level or rangethan in a control sample. Conversely, as used herein with reference toanti-PAD4 autoantibodies, the terms “lower levels,” “low levels,” andvariants thereof refer to a level in a biological sample (e.g., bloodserum) that is lower than a control level or range. Thus, the level ofanti-PAD4 autoantibodies is said to be low or lowered wherein theanti-PAD4 autoantibodies are present in the test sample at a lower levelor range than in a control sample.

The methods disclosed herein can be carried out using any sample thatmay contain anti-PAD4 autoantibodies. Convenient samples include, forexample, whole blood, blood serum, plasma, peripheral blood mononuclearcell samples, or synovial fluid or synovial tissue. In some aspects, asynovial tissue sample can be obtained from a knee joint or anankle-joint. In some aspects, anti-PAD4 autoantibodies are measured in ablood serum sample.

In some aspects, the sample can be pretreated as necessary byconcentration, or dilution in an appropriate buffer solution, such asphosphate, Tris, or the like, at physiological pH.

Anti-PAD4 autoantibodies can be detected and quantified by any of anumber of methods well known to those of skill in the art. These methodsinclude analytic biochemical methods such as electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), hyper diffusion chromatography, massspectroscopy and the like, or various immunological methods such asfluid or gel precipitin reactions, immunodiffusion (single or double),immunohistochemistry, affinity chromatography, immunoelectrophoresis,radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, Western blotting, and the like.

In a specific aspect, anti-PAD4 autoantibodies are detected and/orquantified in the biological sample using an immunoassay. Whileimmunoassays for detecting anti-PAD4 autoantibodies are known in theart, an immunoassay for the detection of anti-PAD4 autoantibodies isdescribed herein.

In certain aspects, the immunoassay comprises a sandwich immunoassay,e.g., an enzyme-linked immunosorbent assay (ELISA) or a sandwichelectrochemiluminescent (ECL) assay, in which a first PAD4 molecule(“capture PAD4”) is attached to a solid support, anti-PAD4autoantibodies from a sample or standard are allowed to bind to thecapture PAD4, and then a second PAD4 molecule (“detection PAD4”) isadded and detected either by an enzymatic reaction, an ECL reaction,radioactivity, or other detection method. Thus, the immunoassay wouldmeasure the anti-PAD4 autoantibody-induced crosslinking of “capturePAD4” and “detection PAD4.” In certain aspects in the immunoassay, thecapture PAD4 molecule is not bound to a solid support.

The term “attached to a solid support” refers to the immobilization thattakes place by attachment to a substrate (e.g., the surface of a well ina plate) by adsorption, covalent binding or by means of a specificbinding pair, e.g., by means of the specific interaction of a suitablespecific binding pair such as biotin/avidin.

The detection PAD4 can be directly labeled with an enzyme, e.g.,horseradish peroxidase or alkaline phosphatase, or can be labeled with atag that will allow an enzyme to bind. Examples of enzyme labels aree.g. alkaline phosphatase, peroxidase and galactosidase. For example thedetection PAD4 can be conjugated to biotin, and the enzyme attached in asubsequent step by allowing enzyme-conjugated streptavidin to bind tothe biotin tag. Alternatively the detection PAD4 can be conjugated to achemiluminescent, fluorescent, or ECL tag. An example of the latter is aruthenium chelate. Following incubation, the plate can then be washed toremove any unbound detection PAD4.

Detection of the detection PAD4 can be accomplished by methods that varybased on the type of detection system that is used. If the detectionPAD4 is tagged with biotin, then enzyme-conjugated streptavidin isadded, unbound streptavidin is washed away, and a substrate is addedwhich provides a colorimetric reaction that can be read, e.g., on aspectrophotometer.

Luminescent metal chelates are metal chelates which generate adetectable luminescence reaction. The detection of this luminescencereaction can for example be by measurement of fluorescence (i.e., if thedetection PAD4 is conjugated to a ruthenium chelate, fluorescence ismeasured) or by electrochemiluminescence (i.e., if the detection PAD4 isconjugated to a ruthenium chelate, the plate can be subjected to anelectrical current, and light emission is measured).

The metal of these metal chelates is for example a transition metal or arare earth metal. The metal can be, for example, ruthenium, osmium,rhenium, iridium, rhodium, platinum, indium, palladium, molybdenum,technetium, copper, chromium or tungsten. The ligands which form themetal chelate together with the metal are usually polydentate ligandsi.e. ligands with several coordination positions. Polydentate ligandsfor example comprise aromatic and aliphatic ligands.

In certain aspects, the method directly measures anti-PAD4 autoantibodylevels in a patient sample, where absolute levels are calculated byplotting the immunoassay results on a standard curve. The detectedsignal can then be quantitated based on the various standards and/orcontrols. By plotting the results on a standard curve, the absolutelevels of anti-PAD4 autoantibody in the samples can be calculated, e.g.,in ng anti-PAD4/mL or anti-PAD4 units/mL (U/mL).

Based on comparison to known control samples, a “threshold level” can bedetermined, and test samples that fall above that anti-PAD4 autoantibodythreshold level can indicate, for example, that the patient from whomthe sample was taken may benefit from treatment with an antibody orantigen-binding fragment thereof that specifically binds to GM-CSFR suchas mavrilimumab. Anti-PAD4 autoantibody threshold levels can bepredetermined, and in that case they must be matched as to the type ofsample (e.g., serum or synovial fluid), the type of RA (e.g.,seropositive or seronegative RA), and in some instances, the assay used.

In some aspects, the predetermined threshold level is the lower limit ofquantification (LLOQ) of the assay used to detect the presence ofanti-PAD4 autoantibodies in a sample. In some aspects, the LLOQ is theLLOQ of the assay presented in Example 1 of the present disclosure. Insome aspects, the LLOQ of the assay used to detect the presence ofanti-PAD4 autoantibodies in a sample is at least 1,000 U/mL, at least1,100 U/mL, at least 1,200 U/mL, at least 1,300 U/mL, at least 1,400U/mL, at least 1,500 U/mL, at least 1,600 U/mL, at least 1,700 U/mL, atleast 1,800 U/mL, at least 1,900 U/mL, at least 2,000 U/mL, at least2,100 U/mL, at least 2,200 U/mL, at least 2,300 U/mL, at least 2,400U/mL, at least 2,500 U/mL, at least 2,600 U/mL, at least 2,700 U/mL, atleast 2,800 U/mL, at least 2,900 U/mL, at least 3,000 U/mL, at least3,100 U/mL, at least 3,200 U/mL, at least 3,300 U/mL, at least 3,400U/mL, at least 3,500 U/mL, at least 3,600 U/mL, at least 3,700 U/mL, atleast 3,800 U/mL, at least 3,900 U/mL, at least 4,000 U/mL, at least4,100 U/mL, at least 4,200 U/mL, at least 4,300 U/mL, at least 4,400U/mL, at least 4,500 U/mL, at least 4,600 U/mL, at least 4,700 U/mL, atleast 4,800 U/mL, at least 4,900 U/mL, at least 5000 U/mL, at least 5500U/mL, at least 6000 U/mL, or at least 6500 U/mL.

In some aspects, the anti-PAD4 autoantibody predetermined thresholdlevel in a sample, e.g., a serum sample, can be at least about 5,000U/mL, at least about 10,000 U/mL, at least about 15,000 U/mL, at leastabout 20,000 U/mL, at least about 25,000 U/mL, at least about 30,000U/mL, at least about 35,000 U/mL, at least about 40,000 U/mL, at leastabout 45,000 U/mL, at least about 50,000 U/mL, at least about 55,000U/mL, at least about 60,000 U/mL, at least about 65,000 U/mL, at leastabout 70,000 U/mL, at least about 75,000 U/mL, at least about 80,000U/mL, at least about 85,000 U/mL, at least about 90,000 U/mL, at leastabout 95,000 U/mL, at least about 100,000 U/mL, at least about 110,000U/mL, at least about 120,000 U/mL, at least about 130,000 U/mL, at leastabout 140,000 U/mL, at least about 150,000 U/mL, at least about 160,000U/mL, at least about 170,000 U/mL, at least about 180,000 U/mL, at leastabout 190,000 U/mL, at least about 200,000 U/mL, at least about 210,000U/mL, at least about 220,000 U/mL, at least about 230,000 U/mL, at leastabout 240,000 U/mL, at least about 250,000 U/mL, at least about 260,000U/mL, at least about 270,000 U/mL, at least about 280,000 U/mL, at leastabout 290,000 U/mL, or at least about 300,000 U/mL as measured in thesample (e.g., serum) using an immunoassay. In some aspects, theimmunoassay is the assay presented in Example 1 of the presentdisclosure.

The anti-PAD4 autoantibody threshold level can vary based on the natureof the assay, e.g., the capture PAD4 and detection PAD4 used, thesource, purity, and composition of the standards, and the like. In oneaspect, instead of using an arbitrary threshold level or the LLOQ of theassay to determine whether a patient can benefit from treatment with anantibody or antigen-binding fragment thereof that specifically binds toGM-CSFRα, for example, mavrilimumab, the patient's anti-PAD4autoantibody levels can be compared to one or more control anti-PAD4autoantibody levels. According to this aspect, the test sample (e.g., asample from a patient suffering from RA) is compared to one or morecontrol samples (e.g., samples taken from normal healthy individuals,earlier samples taken from the same patient, samples taken from patientswith a subset of the patient's disease, a pre-determined standard amountof isolated anti-PAD4 antibodies, or a combination thereof).

In some aspects, the results can be expressed as a ratio with thecontrol samples to determine a variation in the subject's anti-PAD4antibody levels compared to the control levels. According to thisaspect, the control sample can be a matched pair with the patientsample, e.g., one or more of whole blood if the patient sample is wholeblood, serum if the patient sample is serum, plasma if the patientsample is plasma, or synovial fluid if the patient sample is synovialfluid

Anti-PAD4 antibody detection assays can be scored (as positive ornegative or quantity of analyte) according to standard methods wellknown to those of skill in the art. The particular method of scoringwill depend on the assay format and choice of label. For example, animmunoassay can be scored by visualizing a detectably labeled component(e.g., ruthenylated PAD4 in the immunoassay described in Example 1). Adetectable signal (e.g., a luminescent signal above the control orbackground level) is scored as a positive result, while the absence of aclearly detectable signal is scored as a negative and is known as theLLOQ for the assay. The intensity of the detectable signal can provide aquantitative measure of analyte concentration.

Once determined, an anti-PAD4 antibody level can be recorded in apatient medical record. In some aspects, the methods disclosed hereininclude making a diagnosis, often a differential diagnosis, based atleast in part on the anti-PAD4 antibody level.

As used herein, the term “differential diagnosis” can refer to thedetermination of whether a patient is susceptible to treatment, islikely to respond to treatment, or is a candidate for treatment with anantibody or antigen-binding fragment thereof that specifically binds toGM-CSFRα (for example mavrilimumab) depending on whether the measuredanti-PAD4 antibody level in a sample from the patient sample is below apredetermined threshold level, or is low relative to the anti-PAD4antibody level in one or more control samples (e.g., in some aspects apatient may be responsive to mavrilimumab treatment even if the patienttests positive for anti-PAD4 antibodies as long as the level ofanti-PAD4 antibodies is below a predetermined threshold level). Incertain aspects, the predetermined threshold level of anti-PAD4autoantibodies is the LLOQ of the assay.

In particular aspects, the methods disclosed herein include informingthe subject of a result of an assay to detect anti-PAD4 antibodiesand/or of a diagnosis based at least in part on the level of anti-PAD4autoantibodies. The patient can be informed verbally, in writing, and/orelectronically.

This diagnosis can also be recorded in a patient medical record. Forexample, in various aspects, the diagnostic of whether a patient with RAmay benefit by treatment with a specific antibody or antigen-bindingfragment thereof that specifically binds to GM-CSFRα (e.g.,mavrilimumab) is recorded in a medical record. The term “medical record”or “patient medical record” refers to an account of a patient'sexamination and/or treatment that typically includes one or more of thefollowing: the patient's medical history and complaints, the physician'sphysical findings, the results of diagnostic tests and procedures, andpatient medications and therapeutic procedures. A medical record istypically made by one or more physicians and/or physicians' assistantsand it is a written, transcribed or otherwise recorded record and/orhistory of various illnesses or injuries requiring medical care, and/orinoculations, and/or allergies, and/or treatments, and/or prognosis,and/or frequently health information about parents, siblings, and/oroccupation. The record may be reviewed by a physician in diagnosing thecondition.

The medical record can be in paper form and/or can be maintained in acomputer-readable medium. The medical record can be maintained by alaboratory, physician's office, a hospital, a healthcare maintenanceorganization, an insurance company, and/or a personal medical recordwebsite. In some aspects, a diagnosis, based at least in part on thelevel of anti-PAD4 autoantibodies in a sample from a patient, isrecorded on or in a medical alert article such as a card, a wornarticle, and/or a radiofrequency identification (RFID) tag. As usedherein, the term “worn article” refers to any article that can be wornon a subject's body, including, but not limited to, a tag, bracelet,necklace, arm band, or head band.

The methods disclosed herein also include prescribing, initiating,and/or altering prophylaxis and/or therapy for RA (in particular, for asubtype of RA such a seropositive RA or seronegative RA in whichpatients test respectively positive or negative for the presence ofautoantibodies such as anti-PAD4 autoantibodies). In some embodiments,the presence of anti-PAD4 autoantibodies is determined using the methodstaught here.

In certain aspects, the methods can entail ordering and/or performingone or more additional assays. For example, if an assay indicates thatthe patient's sample tests negative for the presence of anti-PAD4autoantibodies, the assay may be repeated to rule out a false negativeresult, and/or one or more additional assays to determine the presenceor absence of anti-PAD4 autoantibodies may be performed to confirm thesubject's status. Conversely, if the levels of anti-PAD4 autoantibodiesare determined to be elevated, it may be desirable repeat the assay torule out a false positive result.

A person skilled in the art would understand that anti-PAD4 autoantibodylevels can be used according to the methods disclosed herein, includingbut not limited to treatment, diagnostic, and monitoring methods, as (i)positive selectors, i.e., a specific action would be taken (e.g.,treating a patient having RA with mavrilimumab) if the level ofanti-PAD4 autoantibodies in a sample taken from the patient is below apredetermined threshold level, below the LLOQ of the detection assayused, or is low relative to the anti-PAD4 autoantibody level in one ormore control samples; or (ii) negative selectors, i.e., a specificaction would be taken (e.g., cease treating a patient having RA withmavrilimumab) if the level of anti-PAD4 autoantibodies in a sample takenfrom the patient is above a predetermined threshold level, above theLLOQ of the detection assay used, or is high relative to the anti-PAD4autoantibody level in one or more control samples; or (iii) bothpositive and negative selectors, for example, a specific treatment couldcease (e.g., DMARD) and a different treatment could commence (e.g.,treatment with mavrilimumab) if the anti-PAD4 autoantibody level in asample taken from the patient is above/below a predetermined thresholdlevel, above/below the LLOQ of the detection assay used, or is high/lowrelative to the anti-PAD4 autoantibody level in one or more controlsamples.

III. Methods of Diagnosis and Treatment RA Based on Anti-PAD4 BiomarkerLevels

This disclosure provides a method of treating an RA patient (or asubject suspected to suffer from RA) comprising administering anantibody or antigen-binding fragment thereof that specifically binds toGM-CSFRα (e.g., mavrilimumab) to the patient if the level of anti-PAD4autoantibodies in a sample taken from the patient is below the lowerlimit of quantification (LLOQ) for the detection assay used (e.g., animmunoassay), below a predetermined threshold level, or low relative tothe anti-PAD4 autoantibody level in one or more control samples. Alsoprovided is method of treating an RA patient (or a subject suspected tosuffer from RA) comprising administering an antibody or antigen-bindingfragment thereof that specifically binds to GM-CSFRα (e.g.,mavrilimumab) to the patient if (a) the level of anti-PAD4autoantibodies in a sample taken from the patient is below the LLOQ forthe detection assay used, below a predetermined threshold level, or lowrelative to the anti-PAD4 autoantibody level in one or more controlsamples and (b) the patient presents a level of at least one additionalbiomarker in a sample taken from the patient which is above or below apredetermined biomarker threshold level, or is above or below thebiomarker level in one or more control samples. In some aspects, themethods further comprise obtaining the sample from the patient andsubmitting the sample for measurement of the level of anti-PAD4autoantibodies in the sample. In one aspect, the presence or absence ofanti-PAD4 autoantibodies in the sample is measured in an immunoassay,e.g., a chemoluminescence assay using PAD4 attached to a substrate anddetectably labeled PAD4 (e.g., ruthenylated PAD4).

This disclosure also provides methods, assays, and kits to facilitate adetermination by a healthcare provider, a healthcare benefits provider,or a clinical laboratory to as to whether a patient will benefit fromtreatment with an antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα (e.g., mavrilimumab). In some aspects,the antibody or antigen-binding fragment thereof that specifically bindsto GM-CSFRα is mavrilimumab, an antibody or fragment thereof that bindsto the same GM-CSFRα epitope as mavrilimumab, or an antibody or fragmentthereof that competitively inhibits binding of mavrilimumab to GM-CSFRα.The methods, assays, and kits provided herein will also facilitate adetermination by a healthcare provider, a healthcare benefits provider,or a clinical laboratory as to whether a patient will benefit fromtreatment with an antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα disclosed herein, or known to those ofordinary skill in the art.

The present disclosure provides methods of treating an RA patient (or asubject suspected to suffer from RA) comprising measuring the level ofanti-PAD4 autoantibodies in a sample (e.g., a blood serum sample)obtained from a patient having RA; determining whether the patient'slevel of anti-PAD4 autoantibodies in the sample is above or below theLLOQ for the assay (e.g., an immunoassay); and advising a healthcareprovider to administer an antibody or antigen-binding fragment thereofthat specifically binds to GM-CSFRα (e.g., mavrilimumab) to the patientif the patient's anti-PAD4 autoantibody level is below the LLOQ for theassay. Conversely, the method would comprise advising a healthcareprovider to suspend the administration of an antibody or antigen-bindingfragment thereof that specifically binds to GM-CSFRα to the patient ifthe patient's anti-PAD4 autoantibody level is above the LLOQ for theassay. In certain embodiments, the anti-PAD4 autoantibody level isdetermined using the assay taught here.

The present disclosure also provides a method of treating an RA patient(or a subject suspected to suffer from RA) comprising submitting asample taken from the patient for measurement of the level of anti-PAD4autoantibodies in the sample; and administering an antibody orantigen-binding fragment thereof that specifically binds to GM-CSFRα(e.g., mavrilimumab) to the patient if the patient's anti-PAD4autoantibody level in the sample is below the LLOQ for the assay. Alsoprovided is a method of treating an RA patient comprising submitting asample taken from the patient for measurement of the level of anti-PAD4autoantibodies in the sample; and suspending the administration of anantibody or antigen-binding fragment thereof that specifically binds toGM-CSFRα (e.g., mavrilimumab) to the patient if the patient's anti-PAD4autoantibody level in the sample is above the LLOQ for the assay.

The present disclosure also provides a method of determining whether totreat a patient diagnosed with RA (or a subject suspected to suffer fromRA) with a therapeutic regimen comprising the administration of anantibody or antigen-binding fragment thereof that specifically binds toGM-CSFRα (e.g., mavrilimumab), comprising: measuring, or instructing aclinical laboratory to measure the level of anti-PAD4 autoantibodies ina sample obtained from the patient diagnosed with RA; and treating, orinstructing a healthcare provider to treat the patient with atherapeutic regimen comprising the administration of an antibody orantigen-binding fragment thereof that specifically binds to GM-CSFRα(e.g., mavrilimumab) if the patient's anti-PAD4 autoantibody level inthe sample is below the LLOQ for the assay. Also provided is a method ofdetermining whether to treat a patient diagnosed with RA with atherapeutic regimen comprising the administration of an antibody orantigen-binding fragment thereof that specifically binds to GM-CSFRα(e.g., mavrilimumab), comprising measuring, or instructing a clinicallaboratory to measure the anti-PAD4 level in a sample obtained from thepatient diagnosed with rheumatoid arthritis; and suspending thetreatment, or instructing a healthcare provider to suspend the treatmentof the patient with a therapeutic regimen comprising the administrationof an antibody or antigen-binding fragment thereof that specificallybinds to GM-CSFRα (e.g., mavrilimumab) if the patient's anti-PAD4autoantibody level in the sample is above the LLOQ for the assay.

The present disclosure also provides methods of selecting a patientdiagnosed with RA (or a subject suspected to suffer from RA) as acandidate for treatment with an antibody or antigen-binding fragmentthereof that specifically binds to GM-CSFRα (e.g., mavrilimumab)comprising measuring, or instructing a clinical laboratory to measurethe level of anti-PAD4 autoantibodies in a sample obtained from apatient diagnosed with RA; and treating, or instructing a healthcareprovider to treat the patient with an antibody or antigen-bindingfragment thereof that specifically binds to GM-CSFRα (e.g.,mavrilimumab) if the patient's anti-PAD4 autoantibody level in thesample is below the LLOQ for the assay. Also provided is method ofselecting a patient diagnosed with RA as a candidate for treatment withan antibody or antigen-binding fragment thereof that specifically bindsto GM-CSFRα (e.g., mavrilimumab) or an alternative RA treatmentcomprising: measuring, or instructing a clinical laboratory to measurethe anti-PAD4 level in a sample obtained from a patient diagnosed withrheumatoid arthritis; and instructing a healthcare provider to suspendthe treatment of the patient with an antibody or antigen-bindingfragment thereof that specifically binds to GM-CSFRα (e.g.,mavrilimumab) or select an alternative treatment if the patient'santi-PAD4 autoantibody level in the sample is above the LLOQ for theassay.

In some aspects of the methods described above, instead of using theLLOQ of the assay as the criterion to make a treatment decision,alternative criteria can be used such as determining whether themeasured levels of anti-PAD4 autoantibodies are above or below apredetermined threshold level, or whether they are high or low relativeto the anti-PAD4 autoantibody level in one or more control samples.

In some aspects, the level of anti-PAD4 autoantibodies in a sampleobtained from an RA patient or a subject suspected to have RA ismeasured in an immunoassay. In certain aspects, the immunoassay isperformed on a sample obtained from the patient or subject suspected tohave RA, by the healthcare professional treating the patient or subject,e.g., using an immunoassay as described herein, formulated as a “pointof care” diagnostic kit. In some aspects, a sample is obtained from thepatient and is submitted, e.g., to a clinical laboratory, formeasurement of the level of anti-PAD4 autoantibodies in the sampleaccording to the healthcare professional's instructions, e.g., using animmunoassay as described herein. In certain aspects, the clinicallaboratory performing the assay will advise the healthcare provide as towhether the patient can benefit from treatment with an antibody orantigen-binding fragment thereof that specifically binds to GM-CSFR(e.g., mavrilimumab) or select an alternative treatment based on whetherthe patient's level of anti-PAD4 autoantibodies is above or below theLLOQ of the immunoassay, above or below a predetermined anti-PAD4autoantibody threshold value or is elevated relative to the level ofanti-PAD4 autoantibodies in one or more control samples.

In certain aspects, this disclosure provides a method of treating an RApatient or a subject suspected of having RA over a period of time,comprising: measuring a first level of anti-PAD4 autoantibodies in afirst sample taken from the patient/subject, or submitting a firstsample taken from the patient/subject for measurement of a first levelof anti-PAD4 autoantibodies in the sample, and administering an antibodyor antigen-binding fragment thereof that specifically binds to GM-CSFR(e.g., mavrilimumab) if the patient's level of anti-PAD4 autoantibodiesis above or below the LLOQ of the immunoassay, above or below apredetermined anti-PAD4 autoantibody threshold value or is elevatedrelative to the level of anti-PAD4 autoantibodies in one or more controlsamples. The test can be performed by a healthcare provider or aclinical laboratory as noted above.

According to this aspect, the method can further comprise: measuring asecond level of anti-PAD4 autoantibodies in a second sample taken fromthe patient, or submitting a second sample taken from the patient formeasurement of a second level of anti-PAD4 autoantibodies in the sample,wherein the patient's level of anti-PAD4 autoantibodies is obtained bycomparing the first and second levels of anti-PAD4 autoantibodies in thepatient, and altering the dose, e.g., increasing or maintaining theamount or frequency of the antibody or antigen-binding fragment thereofthat specifically binds to GM-CSFR (e.g., mavrilimumab) administered tothe patient, or even discontinuing therapy with the antibody orantigen-binding fragment thereof that specifically binds to GM-CSFR(e.g., mavrilimumab) if the patient's anti-PAD4 autoantibody level inthe second sample is higher than the corresponding level in the firstsample, or maintaining or reducing the amount or frequency of theantibody or antigen-binding fragment thereof that specifically binds toGM-CSFR (e.g., mavrilimumab) administered to the patient if thepatient's anti-PAD4 autoantibody level in the second sample is lowerthan or about the same as the corresponding level in the first sample.

In certain aspects, results of an immunoassay as provided herein can besubmitted to a healthcare benefits provider for determination of whetherthe patient's insurance will cover treatment with an antibody orantigen-binding fragment thereof that specifically binds to GM-CSFR(e.g., mavrilimumab).

In certain aspects, the patient has been treated or is being treatedwith one or more additional medications (e.g., DMARDS), either before,during, or after administration of an antibody or antigen-bindingfragment thereof that specifically binds to GM-CSFRα (e.g.,mavrilimumab). Various other medications useful for treating RA aredescribed elsewhere herein or are known in the art. In certain aspectsthe patient has been treated, continues to be treated, or will betreated with one or more additional medications comprising, e.g., aDMARD or a combination thereof. In some aspects, DMARDs are selectedfrom abatacept, adalimumab, azathioprine, chloroquine,hydroxychloroquine, ciclosporin, D-penicillamine, etanercept, golimumab,infliximab, leflunomide, methotrexate, minocycline, rituximab,sulfasalazine, and combinations thereof.

In some aspects, the antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα comprises mavrilimumab or anantigen-binding fragment thereof. In some aspects, the antibody orantigen-binding fragment thereof that specifically binds to GM-CSFRαconsists or consists essentially of mavrilimumab or an antigen-bindingfragment thereof. In some aspects, the antibody or antigen-bindingfragment thereof that specifically binds to GM-CSFRα specifically bindsto the same epitope as mavrilimumab. In some aspects, the antibody orantigen-binding fragment thereof that specifically binds to GM-CSFRαspecifically competes with mavrilimumab for binding to the same epitope.In some aspects, the antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα comprises the heavy chain variable region(VH) comprising the amino acid sequence set forth in SEQ ID NO: 4,and/or the light chain variable region (VL) comprising the amino acidsequence set forth in SEQ ID NO: 5. In some aspects, the antibody orantigen-binding fragment thereof that specifically binds to GM-CSFRαcomprises at least one of the complementarity determining regions whoseamino acid sequence is set forth in SEQ ID NOS: 6-11. In some aspects,the antibody or antigen-binding fragment thereof that specifically bindsto GM-CSFRα comprises the CDRs whose amino acid sequence is set forth inSEQ ID NOS: 6-11.

In some aspects, the samples used in the methods disclosed herein aretaken from a patient and comprise one or more of whole blood, serum,plasma, or synovial fluid.

In some aspects, in addition to the determination of the level ofanti-PAD4 autoantibodies, the methods disclosed herein can furthercomprise determining, submitting a sample taken from the patient fordetermination, or instructing a clinical laboratory to determine theexpression level or activity of one or more biological biomarkers, todetermine one or more clinical biomarkers (clinical status marker), or acombination thereof.

In some aspects, the antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα is administered at a fixed dose. In somespecific aspects, the antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα is mavrilimumab and the fixed dose is atleast 30 mg/dose, at least 100 mg/dose, or at least 150 mg/dose. In someaspects, the antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα, e.g., mavrilimumab, is administered intwo or more doses. In some aspects, the antibody or antigen-bindingfragment thereof that specifically binds to GM-CSFRα, e.g.,mavrilimumab, is administered weekly, biweekly or monthly. In someaspects, the antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα, e.g., mavrilimumab, is administeredintravenously, intramuscularly, subcutaneously, or a combinationthereof.

IV. Anti-PAD4 Autoantibody Detection Assays and Kits

This disclosure also provides kits for detecting anti-PAD4autoantibodies in a sample from a RA patient, for example, through animmunoassay method. Such kits can comprise containers, each with one ormore of the various reagents (e.g., in concentrated form) utilized inthe method, including, for example, labeled and unlabeled PAD4, andanti-PAD4 autoantibody controls.

PAD4 or a fragment thereof capable of binding to anti-PAD4autoantibodies can be provided already attached to a solid support.Labeled PAD4 or a fragment thereof capable of binding to anti-PAD4autoantibodies can be provided already conjugated to a detectable label,e.g., biotin or a ruthenium chelate. The kit can also provide reagentsfor coupling a detectable label to PAD4 or a fragment thereof capable ofbinding to anti-PAD4 autoantibodies (as well as the label itself),buffers, and/or reagents, and instrumentation to support the practice ofthe assays provided herein. A kit provided according to this disclosurecan further comprise suitable containers, plates, and any other reagentsor materials necessary to practice the assays provided herein.

A kit provided according to this disclosure can also comprise brochuresor instructions describing the process. For anti-PAD4 autoantibodydetection immunoassays, the immunoassay process comprises a first PAD4molecule or a fragment thereof that can be recognized by anti-PAD4autoantibodies (“capture PAD4”), and a second PAD4 molecule or afragment thereof that can be recognized by anti-PAD4 autoantibodies,wherein the second PAD4 is detectable, for example, by incorporating afluorescent moiety (“detection PAD4”).

The immunoassay can be performed by methods provided herein or methodswell known and understood by those of ordinary skill in the art. In oneaspect, the immunoassay comprises attaching the capture PAD4 to asupport; applying the test sample or a control sample, allowinganti-PAD4 autoantibodies, if present in the sample, to bind to thecapture PAD4; applying the detection PAD4, which can bind to anti-PAD4autoantibodies already bound to the capture PAD4; and measuring theamount of detection PAD4 bound to anti-PAD4 autoantibodies. In certainaspects, the assay can further include washing steps, blocking steps andincubation steps.

Test kits can include instructions for carrying out one or moreanti-PAD4 autoantibody detection assays, e.g., immunoassays.Instructions included in the kits can be affixed to packaging materialor can be included as a package insert. While the instructions aretypically written or printed materials they are not limited to such. Anymedium capable of storing such instructions and communicating them to anend user is contemplated. Such media include, but are not limited to,electronic storage media (e.g., magnetic discs, tapes, cartridges,chips), optical media (e.g., CD ROM), and the like. As used herein, theterm “instructions” can include the address of an internet site thatprovides the instructions.

V. Computer Methods and Software

The methods disclosed herein can comprise collecting or otherwiseobtaining a biological sample and performing an analytical method todetect and measure anti-PAD4 autoantibody levels alone or in combinationwith other biomarkers, e.g., biological biomarker or clinical biomarker.

Biological biomarkers that can be combined with anti-PAD4 autoantibodylevels include calpastatin, BRAF, C-reactive protein, serum amyloid A,interleukin 6, S100 proteins, osteopontin, rheumatoid factor, matrixmetalloprotease 1, matrix metalloprotease 3, hyaluronic acid, sCD14,angiogenesis markers, products of bone, cartilage, or synoviummetabolism, EYA4, PDZD2, TNF in blood or synovium, IL-1b, IL-17,rheumatoid factor (RF), anticitrullinated peptide antibodies (ACPA), andcombinations thereof. In some aspects, biological biomarkers that can becombined with anti-PAD4 autoantibody levels include TNFRSF17;immunoglobulin J polypeptide, linker protein for immunoglobulin alphaand mu polypeptides (IGJ); POU class 2 associating factor 1 (POU2AF1);Deri-like domain family, member 3 (DERL3); Fc receptor-like 5 (FCRL5);plasma cell-induced ER protein 1 (PACAP); prepronociceptin (PNOC);secreted phosphoprotein 1 (SPP1); interferon regulatory factor 4 (IRF4);lymphocyte transmembrane adaptor 1 (LAX1); ELL associated factor 2(EAF2); pim-2 oncogene (PIM2); actin, alpha 1, skeletal muscle (ACTA1);Myosin, heavy chain 3, skeletal muscle, embryonic (MYH2); myosin, heavychain 1, skeletal muscle, adult (MYH1); cysteine and glycine-richprotein 3 (CSRP3); actinin, alpha 2 (ACTN2); troponin I type 2 (TNNI2);cytochrome P450, family 26, subfamily B, polypeptide 1 (CYP26B1);titin-cap (TCAP), and combinations thereof. See, e.g., U.S. Publ. Nos.US20030027136, US20070231791, which are herein incorporated by referencein their entireties.

Anti-PAD4 autoantibody levels or normalized scores derived from measuredanti-I autoantibody levels can be used alone (e.g., for treatment,diagnostic, prognostic, or monitoring purposes), or in combination withlevels or normalized scores derived from other biomarkers (e.g., a panelof genes used to derive a gene signature). These scores can also becombined with scores corresponding, for example, to clinical biomarkerssuch as (i) gender, (ii) age, (iii) body mass index, (iv) smokingstatus, (v) concomitant drugs, (vi) health assessment quality (HAQ),(vii) disease activity score 28 joints (DAS28), or a combination of twoor more to yield a diagnostic score. In this approach, the diagnosticscore may be a single number determined from the sum of all the markercalculations that is compared to a preset anti-PAD4 autoantibodythreshold value that is an indication of the presence or absence ofdisease. Or the diagnostic score may be a series of bars that eachrepresent a biomarker value and the pattern of the responses may becompared to a pre-set pattern for determination of the presence orabsence of disease.

At least in some aspects of the methods described herein, the complexityof the calculations involved may require that a method comprising theuse of anti-PAD4 autoantibodies as a biomarker be implemented with theuse of a computer. In some aspects, the computer system compriseshardware elements that are electrically coupled via bus, including aprocessor, input device, output device, storage device,computer-readable storage media reader, communications system,processing acceleration (e.g., DSP or special-purpose processors), andmemory. The computer-readable storage media reader can be furthercoupled to computer-readable storage media, the combinationcomprehensively representing remote, local, fixed and/or removablestorage devices plus storage media, memory, etc. for temporarily and/ormore permanently containing computer-readable information, which caninclude storage device, memory and/or any other such accessible systemresource.

A single architecture might be utilized to implement one or more serversthat can be further configured in accordance with currently desirableprotocols, protocol variations, extensions, etc. However, it will beapparent to those skilled in the art that embodiments may well beutilized in accordance with more specific application requirements.Customized hardware might also be utilized and/or particular elementsmight be implemented in hardware, software or both. Further, whileconnection to other computing devices such as network input/outputdevices (not shown) may be employed, it is to be understood that wired,wireless, modem, and/or other connection or connections to othercomputing devices might also be utilized.

In one aspect, the system further comprises one or more devices forproviding input data to the one or more processors. The system furthercomprises a memory for storing a data set of ranked data elements. Inanother aspect, the device for providing input data comprises a detectorfor detecting the characteristic of the data element, e.g., such as afluorescent plate reader, mass spectrometer, or gene chip reader.

The system additionally may comprise a database management system. Userrequests or queries can be formatted in an appropriate languageunderstood by the database management system that processes the query toextract the relevant information from the database of training sets. Thesystem may be connectable to a network to which a network server and oneor more clients are connected. The network may be a local area network(LAN) or a wide area network (WAN), as is known in the art. Preferably,the server includes the hardware necessary for running computer programproducts (e.g., software) to access database data for processing userrequests. The system can be in communication with an input device forproviding data regarding data elements to the system (e.g., expressionvalues). In one aspect, the input device can include a gene expressionprofiling system including, e.g., a mass spectrometer, gene chip orarray reader, and the like.

Some aspects described herein can be implemented so as to include acomputer program product. A computer program product may include acomputer readable medium having computer readable program code embodiedin the medium for causing an application program to execute on acomputer with a database. As used herein, a “computer program product”refers to an organized set of instructions in the form of natural orprogramming language statements that are contained on a physical mediaof any nature (e.g., written, electronic, magnetic, optical orotherwise) and that may be used with a computer or other automated dataprocessing system. Such programming language statements, when executedby a computer or data processing system, cause the computer or dataprocessing system to act in accordance with the particular content ofthe statements.

Computer program products include without limitation: programs in sourceand object code and/or test or data libraries embedded in a computerreadable medium. Furthermore, the computer program product that enablesa computer system or data processing equipment device to act inpre-selected ways may be provided in a number of forms, including, butnot limited to, original source code, assembly code, object code,machine language, encrypted or compressed versions of the foregoing andany and all equivalents.

In one aspect, a computer program product is provided to implement thetreatment, diagnostic, prognostic, or monitoring methods disclosedherein, for example, to determine whether to administer an antibody orantigen-binding fragment thereof that specifically binds to GM-CSFRα(e.g., mavrilimumab) to a patient in need thereof if anti-PAD4autoantibodies are present in a sample taken from the patient, or if thelevel of anti-PAD4 autoantibodies in a sample taken from the patient isabove a predetermined anti-PAD4 autoantibodies threshold level.

The computer program product includes a computer readable mediumembodying program code executable by a processor of a computing deviceor system, the program code comprising:

-   -   code that retrieves data attributed to a biological sample from        a subject, wherein the data comprises anti-PAD4 autoantibodies        level values (presence/absence, amount, or data otherwise        derived from these level values) alone or in combination with        values corresponding to other biomarkers in the biological        sample (e.g., biological markers or clinical markers), and    -   code that executes a classification method that indicates, e.g.,        whether to administer an antibody or antigen-binding fragment        thereof that specifically binds to GM-CSFRα (e.g., mavrilimumab)        to a patient in need thereof.

While various aspects have been described as methods or apparatuses, itshould be understood that aspects can be implemented through codecoupled with a computer, e.g., code resident on a computer or accessibleby the computer. For example, software and databases could be utilizedto implement many of the methods discussed above. Thus, in addition toaspects accomplished by hardware, it is also noted that these aspectscan be accomplished through the use of an article of manufacturecomprised of a computer usable medium having a computer readable programcode embodied therein, which causes the enablement of the functionsdisclosed in this description. Therefore, it is desired that aspectsalso be considered protected by this patent in their program code meansas well.

Furthermore, some aspects can be code stored in a computer-readablememory of virtually any kind including, without limitation, RAM, ROM,magnetic media, optical media, or magneto-optical media. Even moregenerally, some aspects could be implemented in software, or inhardware, or any combination thereof including, but not limited to,software running on a general purpose processor, microcode, PLAs, orASICs.

It is also envisioned that some aspects could be accomplished ascomputer signals embodied in a carrier wave, as well as signals (e.g.,electrical and optical) propagated through a transmission medium. Thus,the various types of information discussed above could be formatted in astructure, such as a data structure, and transmitted as an electricalsignal through a transmission medium or stored on a computer readablemedium.

Specific Embodiments

Embodiment 1: A method of treating a rheumatoid arthritis patientcomprising administering an antibody or antigen-binding fragment thereofthat specifically binds to human granulocyte macrophagecolony-stimulating factor receptor alpha (GM-CSFRα) to the patient ifthe level of anti-peptidylarginine deiminase 4 (anti-PAD4)autoantibodies in a sample taken from the patient is below the lowerlimit of quantification (LLOQ) for the assay.

Embodiment 2: A method of treating a rheumatoid arthritis patientcomprising administering an antibody or antigen-binding fragment thereofthat specifically binds to GM-CSFRα to the patient if the level ofanti-PAD4 in a sample taken from the patient is below the LLOQ for theassay, and the patient presents a level of at least one additionalbiomarker in a sample taken from the patient which is above or below apredetermined biomarker threshold level, or is above or below thebiomarker level in one or more control samples.

Embodiment 3: The method of embodiments 1 or 2, wherein a sample isobtained from the patient and is submitted for measurement of the levelof anti-PAD4 autoantibodies in the sample.

Embodiment 4: A method of treating a rheumatoid arthritis patientcomprising submitting a sample taken from the patient for measurement ofthe anti-PAD4 autoantibody level in the sample; and administering anantibody or antigen-binding fragment thereof that specifically binds toGM-CSFRα to the patient if the patient's anti-PAD4 autoantibody level inthe sample is below the LLOQ for the assay.

Embodiment 5: A method of treating a rheumatoid arthritis patientcomprising submitting a sample taken from the patient for measurement ofthe anti-PAD4 autoantibody level in the sample; and suspending theadministration of an antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα to the patient if the patient's anti-PAD4autoantibody level in the sample is above the LLOQ for the assay.

Embodiment 6: A method of treating a rheumatoid arthritis patientcomprising measuring the anti-PAD4 autoantibody level in a sampleobtained from a patient having rheumatoid arthritis; determining whetherthe patient's anti-PAD4 autoantibody level in the sample is above orbelow the LLOQ for the assay; and advising a healthcare provider toadminister an antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα to the patient if the patient's anti-PAD4autoantibody level is below the LLOQ for the assay.

Embodiment 7: A method of treating a rheumatoid arthritis patientcomprising measuring the anti-PAD4 autoantibody level in a sampleobtained from a patient having rheumatoid arthritis; determining whetherthe patient's anti-PAD4 autoantibody level in the sample is below theLLOQ for the assay; and advising a healthcare provider to suspend theadministration of an antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα to the patient if the patient's anti-PAD4autoantibody level is above the LLOQ for the assay.

Embodiment 8: A method of treating a rheumatoid arthritis patientcomprising submitting a sample taken from the patient for measurement ofthe anti-PAD4 autoantibody level in the sample; and administering anantibody or antigen-binding fragment thereof that specifically binds toGM-CSFRα to the patient if the patient's anti-PAD4 autoantibody level inthe sample is below the LLOQ for the assay.

Embodiment 9: A method of treating a rheumatoid arthritis patientcomprising submitting a sample taken from the patient for measurement ofthe anti-PAD4 autoantibody level in the sample; and suspending theadministration of an antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα to the patient if the patient's anti-PAD4autoantibody level in the sample is above the LLOQ for the assay.

Embodiment 10: A method of determining whether to treat a patientdiagnosed with rheumatoid arthritis with a therapeutic regimencomprising the administration of an antibody or antigen-binding fragmentthereof that specifically binds to GM-CSFRα, comprising: measuring, orinstructing a clinical laboratory to measure the anti-PAD4 autoantibodylevel in a sample obtained from the patient diagnosed with rheumatoidarthritis; and treating, or instructing a healthcare provider to treatthe patient with a therapeutic regimen comprising the administration ofan antibody or antigen-binding fragment thereof that specifically bindsto GM-CSFRα if the patient's anti-PAD4 autoantibody level in the sampleis below the LLOQ for the assay.

Embodiment 11: A method of determining whether to treat a patientdiagnosed with rheumatoid arthritis with a therapeutic regimencomprising the administration of an antibody or antigen-binding fragmentthereof that specifically binds to GM-CSFRα, comprising: measuring, orinstructing a clinical laboratory to measure the anti-PAD4 autoantibodylevel in a sample obtained from the patient diagnosed with rheumatoidarthritis; and suspending the treatment, or instructing a healthcareprovider to suspend the treatment of the patient with a therapeuticregimen comprising the administration of an antibody or antigen-bindingfragment thereof that specifically binds to GM-CSFRα if the patient'santi-PAD4 autoantibody level in the sample is above the LLOQ for theassay.

Embodiment 12: A method of selecting a patient diagnosed with rheumatoidarthritis as a candidate for treatment with an antibody orantigen-binding fragment thereof that specifically binds to GM-CSFRαcomprising: measuring, or instructing a clinical laboratory to measurethe anti-PAD4 autoantibody level in a sample obtained from a patientdiagnosed with rheumatoid arthritis; and treating, or instructing ahealthcare provider to treat the patient with an antibody orantigen-binding fragment thereof that specifically binds to GM-CSFRα ifthe patient's anti-PAD4 level in the sample is below the LLOQ for theassay.

Embodiment 13: A method of selecting a patient diagnosed with rheumatoidarthritis as a candidate for treatment with an antibody orantigen-binding fragment thereof that specifically binds to GM-CSFRαcomprising: measuring, or instructing a clinical laboratory to measurethe anti-PAD4 autoantibody level in a sample obtained from a patientdiagnosed with rheumatoid arthritis; and instructing a healthcareprovider to suspend the treatment of the patient with an antibody orantigen-binding fragment thereof that specifically binds to GM-CSFRα orselect an alternative treatment if the patient's anti-PAD4 autoantibodylevel in the sample is above the LLOQ for the assay.

Embodiment 14: The method according to any one of embodiments 1 to 13,wherein the antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα comprises mavrilimumab or anantigen-binding fragment thereof.

Embodiment 15: The method according to any one of embodiments 1 to 13,wherein the antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα consists of mavrilimumab or anantigen-binding fragment thereof.

Embodiment 16: The method according to any one of embodiments 1 to 13,wherein the antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα specifically binds to the same epitope asmavrilimumab.

Embodiment 17: The method according to any one of embodiments 1 to 13,wherein the antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα specifically competes with mavrilimumabfor binding to the same epitope.

Embodiment 18: The method according to any one of embodiments 1 to 13,wherein the antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα comprises a heavy chain variable regionhaving the amino acid sequence set forth in SEQ ID NO: 4 and/or a lightchain variable region having the amino acid sequence set forth in SEQ IDNO: 5.

Embodiment 19: The method according to any one of embodiments 1 to 13,wherein the antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα comprises at least one of thecomplementarity determining regions having the amino acid sequence setforth in SEQ ID NOS: 6 to 11.

Embodiment 20: The method according to any one of embodiments 1 to 13,wherein the antibody of antigen-binding fragment thereof thatspecifically binds to GM-CSFRα comprises the CDRs having the amino acidsequences set forth in SEQ ID NOs: 6 to 11.

Embodiment 21: The method of any one of embodiments 1 to 20, wherein thepatient has been treated with one or more additional Disease-modifyingantirheumatic drugs (DMARDs), either before, during, or afteradministration of an antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα.

Embodiment 22: The method according to embodiment 21, wherein the DMARDis selected from abatacept, adalimumab, azathioprine, chloroquine,hydroxychloroquine, ciclosporin, D-penicillamine, etanercept, golimumab,infliximab, leflunomide, methotrexate, minocycline, rituximab,sulfasalazine, and combinations thereof.

Embodiment 23: The method according any one of embodiments 1 to 22,wherein the sample obtained from the patient comprises one or more ofwhole blood, blood serum, plasma, or synovial fluid.

Embodiment 24: The method according to embodiment 23, wherein the sampleobtained from the patient is blood serum.

Embodiment 25: The method according to any one of embodiments 1 to 24,further comprising determining, submitting a sample taken from thepatient for determination, or instructing a clinical laboratory todetermine the expression level or activity of one or more additionalbiomarkers, or determine at least one clinical status marker, or acombination thereof.

Embodiment 26: The method according to any one of embodiments 1 to 25,wherein the antibody or antigen-binding fragment thereof thatspecifically binds to GM-CSFRα is administered at a fixed dose.

Embodiment 27: The method according to embodiment 26, wherein theantibody or antigen-binding fragment thereof that specifically binds toGM-CSFRα is administered at a fixed dose of at least 150 mg/dose.

Embodiment 28: The method according to embodiments 1 to 27, wherein thepatient's anti-PAD4 autoantibody level is measured in an immunoassay, anagglutination assay, or a homogeneous assay.

Embodiment 29. The method according to embodiment 28, wherein thepatient's anti-PAD4 autoantibody level is measured in an immunoassay,and the immunoassay employs detectably labeled PAD4.

Embodiment 30: The method according to embodiment 29, wherein thedetectably labeled PAD4 is ruthenylated PAD4.

Embodiment 31: The method according to any one of embodiments 28 to 30,wherein the immunoassay detects anti-PAD4-autoantibody bound to PAD4.

Embodiment 32: An assay for the detection of anti-PAD4 autoantibodiescomprising a first PAD4 molecule or a fragment thereof that can berecognized by anti-PAD4 autoantibodies, and a second PAD4 molecule or afragment thereof that can recognize anti-PAD4 autoantibodies, whereinthe second PAD4 molecule is detectable.

Embodiment 33: The assay according to embodiment 32, wherein afluorescent moiety is incorporated into the detectable PAD4.

Embodiment 34: The assay according to embodiment 33, wherein thefluorescent moiety is ruthenium.

Embodiment 35: A method of treating a rheumatoid arthritis patientcomprising measuring the levels of anti-PAD4 autoantibodies in a samplefrom the patient by using the assay according to any one of embodiments32 to 34, and administering an antibody or antigen-binding fragmentthereof that specifically binds to GM-CSFRα to the patient if the levelof anti-PAD4 autoantibodies in the sample from the patient is below thelower limit of quantification (LLOQ) for the assay.

Embodiment 36: A method of treating a rheumatoid arthritis patientcomprising administering an antibody or antigen-binding fragment thereofthat specifically binds to GM-CSFRα to the patient if the level ofanti-PAD4 in a sample taken from the patient is below the LLOQ for theassay of any one of embodiments 32 to 34, and the patient presents alevel of at least one additional biomarker in a sample taken from thepatient which is above or below a predetermined biomarker thresholdlevel, or is above or below the biomarker level in one or more controlsamples.

Embodiment 37: A method for determining the amount of anti-PAD4autoantibodies in a sample by contacting a capture PAD4 to the testsample or a control sample, allowing anti-PAD4 autoantibodies, ifpresent in the sample, to bind to the capture PAD4; applying a detectionPAD4, which can bind to anti-PAD4 autoantibodies already bound to thecapture PAD4; and measuring the amount of detection PAD4 bound toanti-PAD4 autoantibodies. In certain aspects, the assay can furtherinclude washing steps, blocking steps and incubation steps.

All patents and publications referred to herein are expresslyincorporated by reference in their entireties.

Aspects of the present disclosure can be further defined by reference tothe following non-limiting examples, which describe in detail an assayfor the detection of anti-PAD4 autoantibodies, and use of the assay todetect the presence of anti-PAD4 autoantibodies in clinical samples. Itwill be apparent to those skilled in the art that many modifications,both to materials and methods, can be practiced without departing fromthe scope of the present disclosure.

EXAMPLES Example 1 Anti-PAD4 Autoantibody Assay Protocol

MSD standard plates (available from Meso Scale Discovery) were coatedwith recombinant PAD4 (2 μg/mL in PBS; 50 μL/well; available from CaymanChemical Company, Ann Arbor, MI), and were incubated overnight at 4° C.The plates were washed three times with 200 μL/well wash buffer(PBS/0.05% TWEEN-20®). Following washing, 150 μL/well block buffer(e.g., PBS/0.05% TWEEN-20®/0.2% I-Block Buffer (available from AppliedBiosystems, Carlsbad, CA) was added to each well, and the plates wereincubated for 1-4 hours at room temperature. The plates were then washedthree times as noted above.

For the standard curve, anti-PAD4 autoantibody EIA standard (availablefrom Cayman Chemical Company), was serially diluted in block buffer.Serum samples to be tested for anti-PAD4 autoantibody levels werediluted in block buffer—for example, serum samples from subjects werediluted, e.g., 1:16 in block buffer. Twenty-five microliters (25 μL) ofeach standard or diluted sample was added to the plates, and the plateswere incubated for 1 hour at room temperature with gentle shaking on aplate shaker. Again, the plates were washed three times as noted above.Following washing, ruthenylated recombinant PAD4 (25 μL of a 2 μg/mlsolution) was added to each well, and the plates were incubated for 1hour at room temperature with gentle shaking on a plate shaker. Again,the plates were washed three times as noted above. Following washing,150 L 1×MSD Read Buffer (available from Meso Scale Discovery, Rockville,MD) was added to each well. Finally the plates were read on a MSD platereader (available from Meso Scale Discovery).

Example 2 Detection of Anti-PAD4 Autoantibodies in Serum of RheumatoidArthritis Subjects

This example demonstrates that the anti-PAD4 autoantibody assay providedin this disclosure can be used to detect anti-PAD4 auto-antibodies inthe serum of subjects with rheumatoid arthritis. Serum samples collectedat baseline (at time of study entry) of subjects in three independentclinical trials of mavrilimumab were tested using the anti-PAD4autoantibody assay of Example 1. FIG. 1 shows the measured levels ofanti-PAD4 autoantibodies in all subjects tested from each of the threeclinical trials. A similar frequency of subjects had detectableanti-PAD4 autoantibodies (defined as above the LLOQ for the assay of5000 U/mL) in the three studies, with 32% (Study A), 39% (Study B) and42% (Study C) of subjects testing positive as shown below in TABLE 1.

TABLE 1 Anti-PAD4 Autoantibody Levels in RA Subject Serum Study Status nPercent Mean [Anti-Pad4 Ab] U/mL A Anti-PAD4+ 104 32 268,741 Anti-PAD4−219 68 N/A B Anti-PAD4+ 111 39 214,980 Anti-PAD4− 177 61 N/A CAnti-PAD4+ 57 42 213,881 Anti-PAD4− 80 58 N/A

Example 3 Anti-PAD3 Autoantibody Assay Protocol

MSD standard plates (available from Meso Scale Discovery) were coatedwith recombinant PAD3 (2 μg/mL in PBS; 25 μL/well; available fromSignalChem, Richmond, British Columbia, Canada), and were incubatedovernight at 4° C. The plates were washed three times with 200 μL/wellwash buffer (PBS/0.05% TWEEN-20®). Following washing, 150 μL/well blockbuffer (e.g., PBS/0.05% TWEEN-20®/0.2% I-Block Buffer (available fromApplied BioSystems, Carlsbad, CA) was added to each well, and the plateswere incubated for 1-4 hours at room temperature. The plates were thenwashed three times as noted above.

For the standard, an RA serum sample positive for anti-PAD3 autoantibodywas diluted 1:64 in block buffer. Serum samples to be tested foranti-PAD3 autoantibody levels were diluted in block buffer—for example,serum samples from subjects were diluted, e.g., 1:4 or 1:64 or 1:1024 inblock buffer. Twenty-five microliters (25 μL) of each standard ordiluted sample was added to the plates, and the plates were incubatedfor 1 hour at room temperature with gentle shaking on a plate shaker.Again, the plates were washed three times as noted above. Followingwashing, biotinylated human His10x_Avitag Human PAD3 (25 μL of a 2 μg/mlsolution) was added to each well, and the plates were incubated for 1hour at room temperature with gentle shaking on a plate shaker. Again,the plates were washed three times as noted above. Following washing,streptavidin SulfoTag (15 μL of a 0.5 μg/ml solution) was added to eachwell and incubated for 30 min at room temperature. Again, the plateswere washed three times as noted above. Following washing, 150 μL 1×MSDRead Buffer (available from Meso Scale Discovery, Rockville, MD) wasadded to each well. Finally the plates were read on a MSD plate reader(available from Meso Scale Discovery). Tested samples were determined tobe positive if the detected ECL counts for that sample were above theLLOQ for the assay (a ratio of detected ECL counts in the tested serumsample: ECL counts in a negative control sample ≥1.37).

Detection of Anti-PAD3 Autoantibodies in Serum of Rheumatoid ArthritisSubjects

This example demonstrates that the anti-PAD3 autoantibody assay providedin this disclosure can be used to detect anti-PAD3 auto-antibodies inthe serum of subjects with rheumatoid arthritis. Serum samples collectedat baseline (at time of study entry) of subjects in three independentclinical trials of mavrilimumab were tested using the anti-PAD3autoantibody assay of Example 3. TABLE 2 shows the results of anti-PAD3autoantibody detection in all subjects tested from each of the threeclinical trials. In all three of these studies, a minority of testedsubjects had detectable anti-PAD3 autoantibodies (defined as above theLLOQ for the assay, a ratio of detected ECL counts in the tested serumsample: ECL counts in a negative control sample ≥1.37), with 16% (StudyA), 24% (Study B) and 31% (Study C) of subjects testing positive asshown below in TABLE 2.

TABLE 2 Anti-PAD3 Autoantibody Detection in RA Subject Serum StudyStatus n Percent A Anti-PAD3+ 51 16 Anti-PAD3− 272 84 B Anti-PAD3+ 69 25Anti-PAD3− 211 75 C Anti-PAD3+ 42 31 Anti-PAD3− 94 69

Example 5 A Phase 2b, Randomized, Double-Blind Study to Evaluate theEfficacy and Safety of Mavrilimumab in Subjects with Moderate-to-SevereRheumatoid Arthritis

Study A was a Phase 2b, randomized, double-blind, placebo-controlled,parallel group, multicenter study evaluating the efficacy and safety of3 subcutaneous (SC) doses of mavrilimumab in combination withmethotrexate (MTX) in subjects 18-80 years of age with adult onset RA(defined by the 2010 American College of Rheumatology [ACR] and EuropeanLeague Against Rheumatism [EULAR] classification criteria), withinadequate response to one or more conventional disease-modifyinganti-rheumatic drugs (DMARDs), and at least moderately active disease(defined by Disease Activity Score in 28 joints [DAS28] ≥3.2 C-reactiveprotein [CRP]/erythrocyte sedimentation rate [ESR]) and at least 4swollen joints despite treatment with MTX.

The key inclusion criteria were

-   -   A diagnosis of adult onset RA defined by the 2010 ACR/EULAR        classification criteria (Aletaha et al, 2010)    -   At least moderately active disease as defined by DAS28(CRP)≥3.2        at screening and DAS28(ESR) ≥3.2 at Day 1 (van Gestel et al,        1998)    -   At least 4 swollen joints at screening and Day 1    -   Subjects with inadequate response to one or more conventional        DMARDs    -   Receiving oral or injectable MTX (7.5-25.0 mg/week) for 12 weeks        prior to screening with at least the last 4 weeks prior to        screening at a stable dose

A total of 326 subjects were randomized in a 1:1:1:1 ratio to receiveone of 3 SC doses of mavrilimumab (30, 100, or 150 mg) or placebo Q2Wfor 24 weeks along with a stable dose of oral or injectable MTX (7.525.0 mg/week) as follows:

-   -   Treatment Arm 1: 30 mg SC mavrilimumab Q2W+MTX (n=70)    -   Treatment Arm 2: 100 mg SC mavrilimumab Q2W+MTX (n=70)    -   Treatment Arm 3: 150 mg SC mavrilimumab Q2W+MTX (n=70)    -   Treatment Arm 4: SC placebo Q2W+MTX (n=70)

Subjects received investigational product (mavrilimumab or placebo) bySC injection starting on Week 0 (Day 1) and continued to receiveinvestigational product Q2W for 24 weeks. The primary analysis of thestudy was conducted once the last subject in the study completed theWeek 24 visit. The co-primary endpoints of the study were the changefrom Day 1 DAS28(CRP) score at Week 12 (Day 85) and ACR 20% improvementcriteria (ACR20) at Week 24 (Day 169). The change from baselineDAS28(CRP) at Week 12 (Day 85) was analyzed using a repeated measuresmodel, adjusting for Day 1 DAS28(CRP). Efficacy was evaluated by ACR20,ACR 50% improvement criteria (ACR50), and ACR 70% improvement criteria(ACR70) response rates at each visit using logistic regression.

Subgroup Analysis: Baseline Anti-PAD4 Autoantibodies

To explore the relationship between clinical response to mavrilimumaband peripheral blood biomarkers associated with neutrophil andmacrophage biology, subgroups defined by baseline positivity ornegativity for detectable anti-PAD4 autoantibodies were analyzed.Positivity for anti-PAD4 autoantibodies was defined as having measuredlevels of anti-PAD4 autoantibodies above the LLOQ for the assay.Baseline serum samples were available from 323 of the 326 subjects inthe study and were tested for anti-PAD4 autoantibodies. Overall, 32% ofthe subjects tested positive for anti-PAD4 autoantibodies, with similarpercentages positive in each of the treatment arms as depicted below, onTABLE 3.

TABLE 3 Summary of Anti-PAD4 Autoantibody Analysis Treatment armAnti-PAD4 positive Anti-PAD4 negative Placebo 32% 68% Mavrilimumab 30 mg34% 66% Mavrilimumab 100 mg 29% 71% Mavrilimumab 150 mg 34% 66%

A subgroup analysis was performed for each of the clinical endpoints,including ACR20, ACR50, ACR70 and DAS28(CRP) at the time of the primaryendpoint, after 24 weeks of treatment. Across these clinical endpoints,a significant treatment*biomarker interaction was observed. For theACR20 endpoint, there was a significant treatment*biomarker interaction(p=0.045) such that the anti-PAD4 negative subgroup demonstrated a muchbetter response to mavrilimumab compared to placebo, whereas theanti-PAD4 positive subgroup demonstrated a small difference in responseto mavrilimumab relative to placebo across dose groups (see resultsbelow in TABLE 4). For example, the ACR20 response in the 150 mgmavrilimumab treatment arm in the anti-PAD4 negative subgroup was 77% (Acompared to placebo=60%), while in the anti-PAD4 positive subgroup, theACR20 response was 67% (A compared to placebo=27%).

TABLE 4 ACR20 responses (%) in anti-PAD4 autoantibody positive andnegative subgroups at Week 24 Anti- Anti-PAD4 Anti- Anti-PAD4 TreatmentOverall PAD4 Neg PAD4 Pos Arm Overall (Δ Pbo) Neg (Δ Pbo) Pos (Δ Pbo)Placebo 25 0 17 6 40 0 Mavrilimumab 51 26 45 29 59 19  30 mgMavrilimumab 61 36 65 48 52 12 100 mg Mavrilimumab 73 49 77 60 67 27 150mg

A similar effect was observed for the ACR50 response and again there wasa significant treatment*biomarker interaction (p=0.024). For example,the ACR50 response in the 150 mg mavrilimumab treatment arm in theanti-PAD4 negative subgroup was 40% (A compared to placebo=37%), whilein the anti-PAD4 positive subgroup, the ACR50 response was 41% (Acompared to placebo=9%; see TABLE 56, below).

TABLE 5 ACR50 responses (%) in anti-PAD4 autoantibody positive andnegative subgroups at Week 24 Anti- Anti-PAD4 Anti- Anti-PAD4 TreatmentOverall PAD4 Neg PAD4 Pos Arm Overall (Δ Pbo) Neg (Δ Pbo) Pos (Δ Pbo)Placebo 12 0 4 0 32 0 Mavrilimumab 28 16 23 19 37 5  30 mg Mavrilimumab26 14 28 25 20 −12 100 mg Mavrilimumab 41 28 40 37 41 9 150 mg

A similar effect was observed for the ACR70 response. For example, theACR70 response in the 150 mg mavrilimumab treatment arm in the anti-PAD4negative subgroup was 15% (Δ compared to placebo=15%), while in theanti-PAD4 positive subgroup, the ACR70 response was 11% (Δ compared toplacebo=−1%; TABLE 6). Therefore, the difference in response tomavrilimumab relative to placebo was consistent across ACR20, ACR50 andACR70endpoints. In addition, the difference in response rates in themavrilimumab treatment groups relative to placebo increased in magnitudein the clinical response measures of greater magnitude(i.e. ACR50 andACR70).

TABLE 6 ACR70 responses (%) in anti-PAD4 autoantibody positive andnegative subgroups at Week 24 Anti- Anti-PAD4 Anti- Anti-PAD4 TreatmentOverall PAD4 Neg PAD4 Pos Arm Overall (Δ Pbo) Neg (Δ Pbo) Pos (Δ Pbo)Placebo 4 0 0 0 12 0 Mavrilimumab 12 9 11 11 15 3  30 mg Mavrilimumab 117 10 10 12 0 100 mg Mavrilimumab 14 10 15 15 11 −1 150 mg

The effect of this biomarker was also tested using a fourth measure ofclinical response, the change in DAS28(CRP). A significanttreatment*biomarker interaction (p=0.002) was observed for baselineanti-PAD4 autoantibody levels with the DAS28(CRP) endpoint. For example,the change in DAS28(CRP) in the 150 mg mavrilimumab treatment arm in theanti-PAD4 negative subgroup was −2.15 (Δ compared to placebo=−1.44),while in the anti-PAD4 positive subgroup, the change in DAS28(CRP) was−2.24 (Δ compared to placebo=−0.71). See TABLE 7, below.

TABLE 7 Change in DAS28(CRP) in anti-PAD4 autoantibody positive andnegative subgroups at Week 24 Anti- Anti-PAD4 Anti- Anti-PAD4 TreatmentOverall PAD4 Neg PAD4 Pos Arm Overall (Δ Pbo) Neg (Δ Pbo) Pos (Δ Pbo)Placebo −0.96 0 −0.71 0 −1.53 0 Mavrilimumab −1.74 −0.78 −1.62 −0.91−1.96 −0.43  30 mg Mavrilimumab −1.98 −1.02 −2.14 −1.43 −1.61 −0.08 100mg Mavrilimumab −2.18 −1.22 −2.15 −1.44 −2.24 −0.71 150 mg

Similar trends as those seen with study A were seen with study B.

Subgroup Analysis: Baseline Anti-PAD3 Autoantibodies

To explore the relationship between clinical response to mavrilimumaband autoantibodies to PAD3, a second protein in the protein-argininedeiminase family, subgroups defined by baseline positivity or negativityfor detectable anti-PAD3 autoantibodies were analyzed. Positivity foranti-PAD3 autoantibodies was defined as having measured levels ofanti-PAD3 autoantibodies above the LLOQ for the assay. Baseline serumsamples were available from 323 of the 326 subjects in the study andwere tested for anti-PAD3 autoantibodies. As seen in TABLE 8, below,overall, 16% of the subjects tested positive for anti-PAD3autoantibodies, with similar percentages positive in each of thetreatment arms.

TABLE 8 Summary of Anti-PAD3 Autoantibody Analysis Treatment armAnti-PAD3 positive Anti-PAD3 negative Placebo 16% 84% Mavrilimumab 30 mg11% 89% Mavrilimumab 100 mg 21% 79% Mavrilimumab 150 mg 14% 86%

A subgroup analysis was performed for each of the clinical endpoints,including ACR20, ACR50, ACR70 and DAS28(CRP) at the time of the primaryendpoint, after 24 weeks of treatment. Across these clinical endpoints,no significant treatment*biomarker interaction was observed. As shown inTABLE 9, below, for the ACR20 endpoint, there was no significanttreatment*biomarker interaction such that the anti-PAD3 positive andnegative subgroups demonstrated a similar response to mavrilimumabcompared to placebo. For example, the ACR20 response in the 150 mgmavrilimumab treatment arm in the anti-PAD3 negative subgroup was 74% (Δcompared to placebo=50%), while in the anti-PAD3 positive subgroup, theACR20 response was 73% (Δ compared to placebo=50%).

TABLE 9 ACR20 responses (%) in anti-PAD3 autoantibody positive andnegative subgroups at Week 24 Anti- Anti-PAD3 Anti- Anti-PAD3 TreatmentOverall PAD3 Neg PAD3 Pos Arm Overall (Δ Pbo) Neg (Δ Pbo) Pos (Δ Pbo)Placebo 25 0 24 0 23 0 Mavrilimumab 51 26 48 24 67 44  30 mgMavrilimumab 61 36 63 39 56 33 100 mg Mavrilimumab 73 49 74 50 73 50 150mg

As shown below in TABLE 10, similar effect was observed for the ACR50response and again there was no significant treatment*biomarkerinteraction. For example, the ACR50 response in the 150 mg mavrilimumabtreatment arm in the anti-PAD3 negative subgroup was 40% (Δ compared toplacebo=28%), while in the anti-PAD3 positive subgroup, the ACR50response was 45% (Δ compared to placebo=30%).

TABLE 10 ACR50 responses (%) in anti-PAD3 autoantibody positive andnegative subgroups at Week 24 Anti- Anti-PAD3 Anti- Anti-PAD3 TreatmentOverall PAD3 Neg PAD3 Pos Arm Overall (Δ Pbo) Neg (Δ Pbo) Pos (Δ Pbo)Placebo 12 0 12 0 15 0 Mavrilimumab 28 16 27 15 33 18  30 mgMavrilimumab 26 14 28 16 17 2 100 mg Mavrilimumab 41 28 40 28 45 30 150mg

As shown below, in TABLE 11, a similar effect was observed for the ACR70response. For example, the ACR70 response in the 150 mg mavrilimumabtreatment arm in the anti-PAD3 negative subgroup was 15% (Δ compared toplacebo=10%), while in the anti-PAD3 positive subgroup, the ACR70response was 9% (Δ compared to placebo=9%). Therefore, the difference inresponse to mavrilimumab relative to placebo was consistent acrossACR20, ACR50 and ACR70 endpoints. In addition, the difference inresponse rates in the mavrilimumab treatment groups relative to placebodid not change in magnitude in the clinical response measures of greatermagnitude (i.e. ACR50 and ACR70).

TABLE 11 ACR70 responses (%) in anti-PAD3 autoantibody positive andnegative subgroups at Week 24 Anti- Anti-PAD3 Anti- Anti-PAD3 TreatmentOverall PAD3 Neg PAD3 Pos Arm Overall (Δ Pbo) Neg (Δ Pbo) Pos (Δ Pbo)Placebo 4 0 5 0 0 0 Mavrilimumab 12 9 13 8 11 11  30 mg Mavrilimumab 117 9 4 17 17 100 mg Mavrilimumab 14 10 15 10 9 9 150 mg

The effect of this biomarker was also tested using a fourth measure ofclinical response, the change in DAS28(CRP). As shown below in TABLE 12,no significant treatment*biomarker interaction was observed for baselineanti-PAD3 autoantibody levels with the DAS28(CRP) endpoint. For example,the change in DAS28(CRP) in the 150 mg mavrilimumab treatment arm in theanti-PAD3 negative subgroup was −2.13 (Δ compared to placebo=−1.15),while in the anti-PAD3 positive subgroup, the change in DAS28(CRP) was−2.47 (Δ compared to placebo=−1.73). These results indicate that thetreatment*biomarker interaction obtained for anti-PAD4 antibodies isspecific for anti-PAD4 antibody reactivity and is not a general effectthat extends to all members of the protein-arginine deiminase family.

TABLE 12 Change in DAS28(CRP) in anti-PAD3 autoantibody positive andnegative subgroups at Week 24 Anti- Anti- Anti- PAD3 Anti- PAD3Treatment Overall PAD3 Neg PAD3 Pos Arm Overall (Δ Pbo) Neg (Δ Pbo) Pos(Δ Pbo) Placebo −0.96 0 −0.98 0 −0.74 0 Mavrilimumab −1.74 −0.78 −1.67−0.69 −2.13 −1.39  30 mg Mavrilimumab −1.98 −1.02 −2.07 −1.09 −1.62−0.88 100 mg Mavrilimumab −2.18 −1.22 −2.13 −1.15 −2.47 −1.73 150 mg

It is to be appreciated that the Detailed Description section, and notthe Summary and Abstract sections, is intended to be used to interpretthe claims. The Summary and Abstract sections may set forth one or morebut not all exemplary embodiments of the present invention ascontemplated by the inventor(s), and thus, are not intended to limit thepresent invention and the appended claims in any way.

The present invention has been described above with the aid offunctional building blocks illustrating the implementation of specifiedfunctions and relationships thereof. The boundaries of these functionalbuilding blocks have been arbitrarily defined herein for the convenienceof the description. Alternate boundaries can be defined so long as thespecified functions and relationships thereof are appropriatelyperformed.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance.

The breadth and scope of the present invention should not be limited byany of the above-described exemplary embodiments, but should be definedonly in accordance with the following claims and their equivalents.

TABLE 13 Amino Acid Sequences. SEQ ID NO Sequence Description  1MLLLVTSLLLCELPHPAFLLIPEKSDLRT Granulocyte- VAPASSLNVRFDSRTMNLSWDCQENTTFSmacrophage KCFLTDKKNRVVEPRLSNNECSCTFREIC colony-LHEGVTFEVHVNTSQRGFQQKLLYPNSGR stimulating EGTAAQNFSCFIYNADLMNCTWARGPTAPfactor RDVQYFLYIRNSKRRREIRCPYYIQDSGT receptorHVGCHLDNLSGLTSRNYFLVNGTSREIGI subunit QFFDSLLDTKKIERFNPPSNVTVRCNTTHalpha CLVRWKQPRTYQKLSYLDFQYQLDVHRKN (GM-CSFRα)TQPGTENLLINVSGDLENRYNFPSSEPRA Uniprot: KHSVKIRAADVRILNWSSWSEAIEFGSDDP15509| GNLGSVYIYVLLIVGTLVCGIVLGFLFKR CSF2R_HUMANFLRIQRLFPPVPQIKDKLNDNHEVEDEII WEEFTPEEGKGYREEVLTVKEIT  2QVQLVQSGAEVKKPGASVKVSCKVSGYTL Mavrilimumab TELSIHWVRQAPGKGLEWMGGFDPEENEIheavy chain VYAQRFQGRVTMTEDTSTDTAYMELSSLR (HC)SEDTAVYYCAIVGSFSPLTLGLWGQGTMV TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAP EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGK 3 QSVLTQPPSVSGAPGQRVTISCTGSGSNI MavrilimumabGAPYDVSWYQQLPGTAPKLLIYHNNKRPS light chain GVDRFSGSKSGTSASLAITGLQAEDEADY(LC) YCATVEAGLSGSVFGGGTKLTVLGQPKAA PSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNN KYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS  4 QVQLVQSGAEVKKPGASVKVSCKVSGYTL MavrilimumabTELSIHWVRQAPGKGLEWMGGFDPEENEI heavy chain VYAQRFQGRVTMTEDTSTDTAYMELSSLRvariable SEDTAVYYCAIVGSFSPLTLGLWGQGTMV region (VH) TVS  5QSVLTQPPSVSGAPGQRVTISCTGSGSNI Mavrilimumab GAPYDVSWYQQLPGTAPKLLIYHNNKRPSlight chain GVPDRFSGSKSGTSASLAITGLQAEDEAD variableYYCATVEAGLSGSVFGGGTKLTVL region (VL)  6 YTLTELSIH Mavrilimumab VH-CDR1(VH 27-35)  7 WMGGFDPEENEIVY Mavrilimumab VH-CDR2 (VH 47-60)  8IVGSFSPLTLGL Mavrilimumab VH-CDR3 (VH 98-109)  9 GSNIGAPYDVSMavrilimumab VL-CDR1 (VL 26-36) 10 LLIYHNNKRPS Mavrilimumab VL-CDR2(VL 48-58) 11 ATVEAGLSGS Mavrilimumab VL-CDR3 (VL 91-100) 12 YLDFQMavrilimumab epitope (GM-CSFα 226-230) 13 MAQGTLIRVTPEQPTHAVCVLGTLTQLDIProtein- CSSAPEDCTSFSINASPGVVVDIAHGPPA arginineKKKSTGSSTWPLDPGVEVTLTMKVASGST deiminase GDQKVQISYYGPKTPPVKALLYLTGVEIStype-4 LCADITRTGKVKPTRAVKDQRTWTWGPCG (PAD4)QGAILLVNCDRDNLESSAMDCEDDEVLDS Uniprot: EDLQDMSLMTLSTKTPKDFFTNHTLVLHVQ9UM07| ARSEMDKVRVFQATRGKLSSKCSVVLGPK PADI4_HUMANWPSHYLMVPGGKHNMDFYVEALAFPDTDF PGLITLTISLLDTSNLELPEAVVFQDSVVFRVAPWIMTPNTQPPQEVYACSIFENEDF LKSVTTLAMKAKCKLTICPEEENMDDQWMQDEMEIGYIQAPHKTLPVVFDSPRNRGLK EFPIKRVMGPDFGYVTRGPQTGGISGLDSFGNLEVSPPVTVRGKEYPLGRILFGDSCY PSNDSRQMHQALQDFLSAQQVQAPVKLYSDWLSVGHVDEFLSFVPAPDRKGFRLLLAS PRSCYKLFQEQQNEGHGEALLFEGIKKKKQQKIKNILSNKTLREHNSFVERCIDWNRE LLKRELGLAESDIIDIPQLFKLKEFSKAEAFFPNMVNMLVLGKHLGIPKPFGPVINGR CCLEEKVCSLLEPLGLQCTFINDFFTYHIRHGEVHCGTNVRRKPFSFKWWNMVP

1-20. (canceled)
 21. A method of treating a rheumatoid arthritis patientcomprising identifying the rheumatoid arthritis patient havinganti-peptidylarginine deiminase 4 (anti-PAD4) autoantibody level belowthe lower limit of quantification (LLOQ) for an assay of 5000 U/ml; andadministering an antibody or antigen-binding fragment thereof thatinhibits association between human granulocyte macrophagecolony-stimulating factor receptor alpha (GM-CSFRα) and its ligandGM-CSF to the patient.
 22. The method of claim 21, wherein the antibodyor antigen binding fragment thereof that inhibits association betweenGM-CSFRα and its ligand GM-CSF specifically binds to GM-CSFRα.
 23. Themethod of claim 21, wherein the antibody or antigen-binding fragmentthereof that inhibits association between GM-CSFRα and its ligand GM-CSFcomprises complementary determining regions having the amino acidsequence set forth in SEQ ID NOS: 6 to
 11. 24. The method of claim 21,wherein the antibody or antigen-binding fragment thereof that inhibitsassociation between GM-CSFRα and its ligand GM-CSF comprises a heavychain variable region having the amino acid sequence set forth in SEQ IDNO: 4 and a light chain variable region having the amino acid sequenceset forth in SEQ ID NO:
 5. 25. The method of claim 21, wherein thepatient has been treated with one or more additional Disease-modifyingantirheumatic drugs (DMARDs), either before, during, or afteradministration of an antibody or antigen-binding fragment thereof thatinhibits association between GM-CSFRα and its ligand GM-CSF.
 26. Themethod claim 21, wherein the anti-PAD4 autoantibody level is detected inone or more of the patient's whole blood, blood serum, plasma, orsynovial fluid.
 27. The method according to claim 6, wherein theanti-PAD4 autoantibody level is detected in the patient's blood serum.28. The method of claim 21, further comprising a step of determining,submitting a sample taken from the patient for determination, orinstructing a clinical laboratory to determine the expression level oractivity of one or more additional biomarkers, or to determine at leastone clinical status marker, or a combination thereof.
 29. The method ofclaim 21, wherein the antibody or antigen-binding fragment thereof thatinhibits association between GM-CSFRα and its ligand GM-CSF isadministered at a fixed dose.
 30. The method of claim 21, wherein theassay is an immunoassay, an agglutination assay, or a homogeneous assay.31. The method according to claim 30, wherein the assay is animmunoassay, and the immunoassay employs detectably labeled PAD4. 32.The method according to claim 31, wherein the detectably labeled PAD4 isruthenylated PAD4.
 33. The method of claim 30, wherein the immunoassaydetects anti-PAD4-autoantibody bound to PAD4.